Lipoxygenase proteins and nucleic acids

ABSTRACT

Isolated and purified lipoxygenase proteins and nucleic acids are described. Particularly, a novel human 15(S) lipoxygenase (15-Lox-2) protein and cDNA and a cDNA for mouse 8S-lipoxygenase are described. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also described, along with methods of producing each. Isolated and purified antibodies to 15-Lox-2 and 8-Lox, and methods of producing the same, are also described.

GRANT STATEMENT

[0001] This work was supported by NIH grants GM-53638 and GM-49502; andPilot Project grants from the Vanderbilt Skin Disease Research Center(SDRC) from grant 5P30 AR41943-03 from the NIH/NIAMS and from the Centerin Molecular Toxicology (USPHS ES000267). The U.S. Government hascertain rights in the invention.

TECHNICAL FIELD

[0002] The present invention relates generally to isolated and purifiedlipoxygenase proteins and nucleic acids. More particularly, the presentinvention relates to an isolated and purified second type of human15S-lipoxygenase and an isolated and purified nucleic acid encoding thesame, and to an isolated and purified nucleic acid encoding a mouse8S-lipoxygenase. Table of Abbreviations 15-Lox-1 Reticulocyte-type of15S-lipoxygenase 15-Lox-2 Second type of human 15S-lipoxygenase 8-Loxmouse 8S-lipoxygenase PMA Phorbol-12-myristate-13-acetate H(P)ETEHydro(pero)xyeicosatetraenoic acid HODE Hydroxyoctadecadienoic acid HPLCHigh pressure liquid chromatography PCR Polymerase chain reaction RACERapid amplification of cDNA ends

BACKGROUND ART

[0003] The lipoxygenases are a structurally related family of non-hemeiron dioxygenases that function in the production of fatty acidhydroperoxides. Three lipoxygenases have been identified and cloned inhumans. Funk, C. D. (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98;Matsumoto et al. (1988) Proc. Natl. Acad. Sci. USA 85: 26-30; Dixon etal. (1988) Proc. Natl. Acad. Sci. USA 85: 416-420; Funk et al. (1990)Proc. Natl. Acad. Sci. USA 87: 5638-5642; Izumi et al. (1990) Proc.Natl. Acad. Sci. USA 87:7477-7481; Yoshimoto et al. (1990) Biochem.Biophys. Res. Comm. 172:1230-1235; Sigal et al. (1988) Biochem. Biophys.Res. Comm. 157:457-464). They oxygenate arachidonic acid in differentpositions along the carbon chain and form the corresponding 5S-, 12S- or15S-hydroperoxides (hydroperoxy-eicosatetraenoic acids, HPETEs). Thethree enzymes are known mainly from the blood cell types in which theyare strongly expressed—the 5S-lipoxygenase of leukocytes, the12S-lipoxygenase of platelets, and the 15S-lipoxygenase ofreticulocytes, eosinophils and macrophages. While these are the mostwidely recognized cellular sources, selective expression is welldocumented in other tissues. For example, both the 12S- and15S-lipoxygenases are detected in skin. Nugteren et al. (1987) Biochim.Biophys. Acta 921:135-141; Henneicke-von Zepelin et al. (1991) J.Invest. Dermatol. 97:291-297; Takahashi et al. (1993) J. Biol. Chem.268:16443-16448;Hussain et al. (1994) Amer. J. Physiol. 266:C243-C253.

[0004] Potentially, the three cloned lipoxygenases could account for allenzymatic synthesis of arachidonate hydroperoxides in humans, but thereare reasons to consider that other lipoxygenases may exist. For example,in the mouse there are five known lipoxygenases, three that correspondto the known human enzymes, Chen et al. (1994) J. Biol. Chem.269:13979-13987; Chen et al. (1995) J. Biol. Chem. 270:17993-17999 andtwo others, Furstenberger et al. (1991) J. Biol. Chem. 266:15738-15745;Funk et al. (1996) J. Biol. Chem. 271:23338-23344.

[0005] Three of the five distinct mouse lipoxygenase enzymes are bestknown for their occurrence in different types of blood cells. In commonwith other mammals, a 5S-lipoxygenase is present in leukocytes and isresponsible for synthesis of the pro-inflammatory mediators, theleukotrienes. Chen et al. (1995) J. Biol. Chem. 270:17993-17999; Chen etal. (1994) Nature 372:179-182. A 12S-lipoxygenase is found in plateletsand several other tissues including skin. Nugteren et al. (1987)Biochim. Biophys. Acta 921:135-141; Chen et al. (1994) J. Biol. Chem.269:13979-13987; Sun et al. (1996) J. Biol. Chem. 271:24055-24062.

[0006] A second type of 12S-lipoxygenase which is closely related insequence to the human and rabbit “reticulocyte-type” of15S-lipoxygenases occurs in certain macrophages. Sun et al. (1996) J.Biol. Chem. 271, 24055-24062. The fourth mouse lipoxygenase to becharacterized is another enzyme to have 12S-lipoxygenase activity; itwas cloned recently from mouse skin and has been classified as anepidermal 12S-lipoxygenase. van Dijk et al. (1995) Biochim. Biophys.Acta 1259:4-8; Funk et al. (1996) J. Biol. Chem. 271:23338-23344. Allfour of these murine lipoxygenases enzymes have been characterized atthe cDNA and genomic levels.

[0007] The fifth known mouse lipoxygenase was described originally in1986 by Furstenberger, Marks and colleagues as an enzyme in skin forming8-HETE and inducible by phorbol ester treatment. Gschwendt et al. (1986)Carcinogenesis 7:449-455. It was shown subsequently that this enzymeforms the 8S enantiomer (Hughes et al. (1991) Biochim. Biophys. Acta1081:347-354) and isolation of the corresponding hydroperoxide confirmedidentification of the enzyme as a lipoxygenase. Fürstenberger et al.(1991) J. Biol. Chem. 266:15738-15745. Mouse skin is the only reportedsite of synthesis of 8S-HETE in animal tissues, and there is noindication from the literature pointing to a potential homologue of themouse 8S-lipoxygenase in other mammals. Additionally, no nucleic acid,particularly a cDNA, which encodes this lipoxygenase has beencharacterized.

[0008] Despite the description in the art of the enzymes presentedabove, along with the catalytic activities covered by these enzymes,there remains an open question whether a lipoxygenase rather than acytochrome P450 might account for the synthesis of 12R-hydroxyarachidonic acid (12R-HETE), Hammarström et al. (1975) Proc. Natl. Acad.Sci. USA 72:5130-5134; Woollard, P. M. (1986) Biochem. Biophys. Res.Commun. 136(1):169-175; Baer et al. (1991) J. Lipid Research 32:341-347;Holtzman et al. (1989) J. Clin. Invest. 84:1446-1453; Brash et al.(1996) J. Biol. Chem. 271:20549-20557, a prominent arachidonatemetabolite in the skin disease of psoriasis and other proliferativedermatoses (Hammarström et al. (1975) Proc. Natl. Acad. Sci. USA72:5130-5134; Baer et al. (1991) J. Lipid Research 32:341-347; Baer etal. (1995) J. Invest. Dermatol. 104:251-255).

[0009] Therefore, what is needed, then, is further characterization oflipoxygenase enzymes in vertebrates, particularly in mammals, and moreparticularly in humans. A novel isolated and purified lipoxygenase and anucleic acid encoding the same would have broad utility to due its rolein arachidonic acid metabolism, a critical metabolic pathway.

DISCLOSURE OF THE INVENTION

[0010] A key aspect of this invention pertains to the discovery of anovel 15S-lipoxygenase (15-Lox-2) protein and nucleic acid encoding the15-Lox-2 protein. Preferred nucleic acid and amino acid sequences for15-Lox-2 are described in SEQ ID NO:1 and SEQ ID NO:2.

[0011] It is another aspect of this invention that the novel 15-Lox-2protein acts in the metabolism of arachidonic acid to15S-Hydro(pero)xyeicosatetraenoic acid.

[0012] Another key aspect of this invention is isolation andpurification of a nucleic acid encoding mouse 8S-lipoxygenase (8-Lox) Apreferred embodiment of this nucleic acid is described in SEQ ID NO:3.

[0013] Thus, in one aspect, the present invention provides an isolatedand purified polynucleotide that encodes a lipoxygenase polypeptidewherein the lipoxygenase polypeptide includes an iron ligand comprisinga serine. Preferably, the lipoxygenase polypeptide reacts witharachidonic acid. In a preferred embodiment, a polynucleotide of thepresent invention is a DNA molecule from a vertebrate species. Apreferred vertebrate is a mammal. A preferred mammal is a human. Morepreferably, a polynucleotide of the present invention encodespolypeptides designated 15-Lox-2 and 8-Lox. Even more preferred, apolynucleotide of the present invention encodes a polypeptide comprisingthe amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4. Mostpreferably, an isolated and purified polynucleotide of the inventioncomprises the nucleotide base sequences of SEQ ID NO:1 or SEQ ID NO:3 ortheir homologues from other vertebrate species.

[0014] Yet another aspect of the present invention contemplates anisolated and purified polynucleotide comprising a base sequence that isidentical or complementary to a segment of at least 10 contiguous basesof SEQ ID NO:1 wherein the polynucleotide hybridizes to a polynucleotidethat encodes a lipoxygenase polypeptide wherein the lipoxygenasepolypeptide includes an iron ligand comprising a serine. Preferably, thelipoxygenase polypeptide reacts with arachidonic acid. Preferably, theisolated and purified polynucleotide comprises a base sequence that isidentical or complementary to a segment of at least 25 to 70 contiguousbases of SEQ ID NO:1. For example, a polynucleotide of the invention cancomprise a segment of bases identical or complementary to 40 or 55contiguous bases of the disclosed nucleotide sequences.

[0015] In another embodiment, the present invention contemplates anisolated and purified lipoxygenase polypeptide wherein the lipoxygenasepolypeptide includes an iron ligand comprising a serine. Preferably, thelipoxygenase polypeptide reacts with arachidonic acid. More preferably,a polypeptide of the invention is a recombinant polypeptide. Even morepreferably, a polypeptide of the present invention is 15-Lox-2. Evenmore preferably, a polypeptide of the present invention comprises theamino acid residue sequence of SEQ ID NO:2.

[0016] In an alternative embodiment, the present invention provides anexpression vector comprising a polynucleotide that encodes alipoxygenase polypeptide that includes an iron ligand comprising aserine. Preferably, the lipoxygenase polypeptide reacts with arachidonicacid. Also preferably, an expression vector of the present inventioncomprises a polynucleotide that encodes 15-Lox-2 or 8-Lox. Morepreferably, an expression vector of the present invention comprises apolynucleotide that encodes a polypeptide comprising the amino acidresidue sequence of SEQ ID NO:2 or SEQ ID NO:4. More preferably, anexpression vector of the present invention comprises a polynucleotidecomprising the nucleotide base sequence of SEQ ID NO:1 or SEQ ID NO:3.Even more preferably, an expression vector of the invention comprises apolynucleotide operatively linked to an enhancer-promoter. Morepreferably still, an expression vector of the invention comprises apolynucleotide operatively linked to a prokaryotic promoter.Alternatively, an expression vector of the present invention comprises apolynucleotide operatively linked to an enhancer-promoter that is aeukaryotic promoter, and the expression vector further comprises apolyadenylation signal that is positioned 3′ of the carboxy-terminalamino acid and within a transcriptional unit of the encoded polypeptide.

[0017] In yet another embodiment, the present invention provides arecombinant host cell transfected with a polynucleotide that encodes alipoxygenase polypeptide which includes an iron ligand comprising aserine. Preferably, the lipoxygenase polypeptide reacts with arachidonicacid. SEQ ID NO:1; SEQ ID NO: 2 SEQ ID NO:3; and SEQ ID NO: 4 set forthnucleotide and amino acid sequences from the exemplary vertebrates humanand mouse. Also contemplated by the present invention are homologous orbiologically equivalent polynucleotides and lipoxygenase polypeptidesfound in other vertebrates. Preferably, a recombinant host cell of thepresent invention is transfected with the polynucleotide that encodes15-Lox-2 or 8-Lox. More preferably, a recombinant host cell of thepresent invention is transfected with the polynucleotide sequence of SEQID NO:1 or SEQ ID NO:3. Even more preferably, a host cell of theinvention is a eukaryotic host cell. Still more preferably, arecombinant host cell of the present invention is a vertebrate cell.Preferably, a recombinant host cell of the invention is a mammaliancell.

[0018] In another aspect, a recombinant host cell of the presentinvention is a prokaryotic host cell. Preferably, a recombinant hostcell of the invention is a bacterial cell, preferably a strain ofEscherichia coli. More preferably, a recombinant host cell comprises apolynucleotide under the transcriptional control of regulatory signalsfunctional in the recombinant host cell, wherein the regulatory signalsappropriately control expression of a lipoxygenase polypeptide thatmetabolizes arachidonic acid in a manner to enable all necessarytranscriptional and post-transcriptional modification.

[0019] In yet another embodiment, the present invention contemplates aprocess of preparing a lipoxygenase polypeptide comprising transfectinga cell with polynucleotide that encodes a lipoxygenase polypeptide whichincludes an iron ligand comprising a serine, to produce a transformedhost cell; and maintaining the transformed host cell under biologicalconditions sufficient for expression of the polypeptide. Preferably, thelipoxygenase polypeptide that is produced reacts with arachidonic acid.More preferably, the transformed host cell is a eukaryotic cell. Morepreferably still, the eukaryotic cell is a vertebrate cell.Alternatively, the host cell is a prokaryotic cell. More preferably, theprokaryotic cell is a bacterial cell of the DH5α strain of Escherichiacoli. Even more preferably, a polynucleotide transfected into thetransformed cell comprises the nucleotide base sequence of SEQ ID NO:1or SEQ ID NO:3. SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4set forth nucleotide and amino acid sequences for the exemplaryvertebrates human and mouse. Also contemplated by the present inventionare homologues or biologically equivalent lipoxygenase polynucleotidesand polypeptides found in other vertebrates.

[0020] In still another embodiment, the present invention provides anantibody immunoreactive with a lipoxygenase polypeptide which includesan iron ligand comprising a serine. Preferably, the lipoxygenasepolypeptide reacts with arachidonic acid. SEQ ID NO:1; SEQ ID NO:2; SEQID NO:3; and SEQ ID NO:4 set forth nucleotide and amino acid sequencesfrom the exemplary vertebrates human and mouse. Also contemplated by thepresent invention are antibodies immunoreactive with homologues orbiologically equivalent lipoxygenase polynucleotides and polypeptidesfound in other vertebrates. Preferably, an antibody of the invention isa monoclonal antibody. More preferably, the lipoxygenase polypeptidecomprises 15-Lox-2 or 8-Lox. Even more preferably, a polypeptidecomprises the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0021] In another aspect, the present invention contemplates a processof producing an antibody immunoreactive with a lipoxygenase polypeptidewhich includes an iron ligand comprising a serine, the processcomprising the steps of (a) transfecting a recombinant host cell with apolynucleotide that encodes a lipoxygenase polypeptide which includes aniron ligand comprising a serine; (b) culturing the host cell underconditions sufficient for expression of the polypeptide; (c) recoveringthe polypeptide; and (d) preparing the antibody to the polypeptide.Preferably, the lipoxygenase polypeptide reacts with arachidonic acid.SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4 set forthnucleotide and amino acid sequences from the exemplary vertebrates mouseand human. Preferably, the host cell is transfected with thepolynucleotide of SEQ ID NO:1 or SEQ ID NO:3. Even more preferably, thepresent invention provides an antibody prepared according to the processdescribed above. Also contemplated by the present invention is the useof homologues or biologically equivalent polynucleotides andpolypeptides found in other vertebrates to produce antibodies.

[0022] Alternatively, the present invention provides a process ofdetecting a lipoxygenase polypeptide that metabolizes arachidonic acid,wherein the process comprises immunoreacting the polypeptide with anantibody prepared according to the process described above to form anantibody-polypeptide conjugate, and detecting the conjugate.

[0023] In yet another embodiment, the present invention contemplates aprocess of detecting a messenger RNA transcript that encodes alipoxygenase polypeptide which includes an iron ligand comprising aserine, wherein the process comprises hybridizing the messenger RNAtranscript with a polynucleotide sequence that encodes that polypeptideto form a duplex; and detecting the duplex. Alternatively, the presentinvention provides a process of detecting a DNA molecule that encodes alipoxygenase polypeptide, wherein the process comprises hybridizing DNAmolecules with a polynucleotide that encodes a lipoxygenase polypeptidewhich includes an iron ligand comprising a serine to form a duplex; anddetecting the duplex. For both such processes, it is preferred that thedetected lipoxygenase polypeptide is capable of reacting witharachidonic acid.

[0024] In another aspect, the present invention contemplates adiagnostic assay kit for detecting the presence of a lipoxygenasepolypeptide in a biological sample, where the kit comprises a firstcontainer containing a first antibody capable of immunoreacting with alipoxygenase polypeptide which includes an iron ligand comprising aserine, with the first antibody present in an amount sufficient toperform at least one assay. Preferably, an assay kit of the inventionfurther comprises a second container containing a second antibody thatimmunoreacts with the first antibody. More preferably, the antibodiesused in an assay kit of the present invention are monoclonal antibodies.Even more preferably, the first antibody is affixed to a solid support.More preferably still, the first and second antibodies comprise anindicator, and, preferably, the indicator is a radioactive label or anenzyme.

[0025] In an alternative aspect, the present invention provides adiagnostic assay kit for detecting the presence, in biological samples,of a lipoxygenase polypeptide, the kits comprising a first containerthat contains a second polynucleotide identical or complementary to asegment of at least 10 contiguous nucleotide bases of a polynucleotidethat encodes a lipoxygenase polypeptide which includes an iron ligandcomprising a serine. Preferably, the polynucleotide encodes alipoxygenase polypeptide capable of reacting with arachidonic acid. Morepreferably, the polynucleotide encodes 15-Lox-2 or 8-Lox.

[0026] In another embodiment, the present invention contemplates adiagnostic assay kit for detecting the presence, in a biological sample,of an antibody immunoreactive with a lipoxygenase polypeptide, the kitcomprising a first container containing a lipoxygenase polypeptide whichincludes an iron ligand comprising a serine that immunoreacts with theantibody, with the polypeptide present in an amount sufficient toperform at least one assay. Preferably, the lipoxygenase polypeptide iscapable of reacting with arachidonic acid. More preferably, thepolypeptide comprises 15-Lox-2 or 8-Lox.

[0027] The foregoing aspects and embodiments have broad utility giventhe biological significance of the arachidonic acid pathway, as is knownin the art. By way of example, the foregoing aspects and embodiments areuseful in the preparation of screening assays and assay kits that areused to identify compounds that affect arachidonic acid metabolism, orthat are used to detect the presence of the proteins and nucleic acidsof this invention in biological samples. Additionally, it is well knownthat isolated and purified polypeptides have utility as feed additivesfor livestock and further polynucleotides encoding the polypeptides arethus useful in producing the polypeptides.

[0028] Following long-standing patent law convention, the terms “a” and“an” mean “one or more” when used in this application, including theclaims.

[0029] Some of the aspects and objects of the invention having beenstated hereinabove, other aspects and objects will become evident as thedescription proceeds, when taken in connection with the accompanyingdrawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 shows sequence alignment of human 15S-lipoxygenases. Thetop line shows the amino acid sequence (SEQ ID NO:2) deduced from thenew human lipoxygenase (15-Lox-2) cDNA, in alignment with the sequenceof the previously reported human 15S-lipoxygenase (15-Lox-1) (SEQ IDNO:25). Sigal et al. (1988) Biochem. Biophys. Res. Comm. 157: 457-464.The consensus sequences used in PCR cloning are underlined, and fiveputative iron ligands (H374, H379, H554, S558, I676-15-Lox-2; H374,H379, H554, H558, I676-15-Lox-1) are in bold. Two clones of the new CDNAwere sequenced: there was a single nucleotide difference (position 1263in the open reading frame, C or T) which did not change the deducedamino acid sequence. The new cDNA sequence (SEQ ID NO:1) is available inthe GenBank™/EMBL Data Bank with accession number U78294.

[0031]FIG. 2 shows expression in HEK 293 cells: identification of the15S-HETE product. Following transient expression of the cDNA, the HEK293 cells were sonicated in 50 mM Tris (pH 7.5) containing 100 mM NaCl,then incubated with [¹⁴C]arachidonic acid (50 μM) for 30 min at 37° C.,and the products extracted as described IN Brash et al. (1996) J. Biol.Chem. 271:20549-20557.

[0032]FIG. 2A shows reversed-phase HPLC analysis of the products using aBeckman 5μ ODS Ultrasphere column (25×0.46 cm) with a Bio-Rad 5S ODSguard column, a solvent system of methanol:water:glacial acetic acid(80:20:0.01, by volume), and a flow rate of 1.1 ml/min with on-linedetection of radiolabeled products using a Packard Flo-One Radiomaticdetector. Retention times of HETE standards are indicated onchromatogram. The small peak on the front shoulder of the 15-HETE is15-keto-eicosatetraenoic acid.

[0033]FIG. 2B shows chiral analysis of the methyl ester derivative ofthe 15-HETE product using a Chiralcel OB column with a solvent ofhexane:isopropanol (100:2, v/v) and a flow rate of 1.1 ml/min.

[0034]FIGS. 3A and 3B shows multiple human tissue RNA blots. Two tissueblots of mRNA (Clontech) were probed with a 1067 bp fragment of the newhuman lipoxygenase cDNA.

[0035]FIG. 4 shows detection of the new 15S-lipoxygenase transcript inhuman cornea.

[0036]FIG. 4A depicts RT-PCR wherein RNA samples were treated with DNAse1, then reverse transcribed to cDNA. PCR reactions were run using twoprimer sets (1 and 2, see Experimental Procedures of Example 1) withhuman cornea cDNA as template, and also with rabbit cornea cDNA andbuffer (H₂O) alone as negative controls. Bands of the correct sizes, 589bp and 351 bp, are detected in human cornea; the larger band wassubcloned and sequenced, confirming the identity to the lipoxygenasecDNA cloned from skin.

[0037]FIG. 4B depicts a Northern analysis of human eye tissues. The bandin cornea mRNA at about 2.5-3 KB corresponds to the new lipoxygenasetranscript.

[0038]FIG. 5 shows nucleotide (SEQ ID NO:3) and deduced amino acid (SEQID NO:4) sequences of the mouse 8S lipoxygenase. The sequence is fromtwo identical cDNA library clones, except for the 5′ UTR, which wasobtained by 5′ RACE. The consensus sequences used in PCR cloning areunderlined. Seven PCR clones encoding the open reading frame and whichexpressed 8S-lipoxygenase activity were also fully sequenced. Thesecontained multiple nucleotide substitutions which changed the encodedamino acid sequence yet had no apparent detrimental effect on theirexpressed 8S-lipoxygenase activity: clone #G2, 112A (Leu to Met), 227-C(Val to Ala), 1607-A (Arg to Gln) clone #G5, 227-C (Val to Ala), 1607-A(Arg to Gln); clone #G11, 227-C (Val to Ala), 1607-A (Arg to Gln); clone#K1, 227-C (Val to Ala); clone #K2, same amino acid sequence as libraryclone; clone #K7, 1237-G (Ile to Val); clone #K12, 95-G (Glu to Gly),173-G (Pro to Arg). The cDNA sequence is available in GenBank withaccession no. U93277.

[0039]FIG. 6 shows alignment of mouse 8S-lipoxygenase with the secondtype of human 15S-lipoxygenase (15-Lox-2). The top line shows the aminoacid sequence (SEQ ID NO:4) deduced from the mouse 8S-lipoxygenase cDNA,in alignment with the amino acid sequence (SEQ ID NO:2) of the secondtype of human 15S-lipoxygenase. Five putative iron ligands are inboldface (H374,H379,H554, S558, I676-8-Lox; H374, H379, H554, S558,I676-15-Lox-2).

[0040]FIG. 7 shows expression of 8S-lipoxygenase in vaccinia virusinfected Hela cells. Cells sonicates were incubated with[1-¹⁴C]arachidonic acid (100 μM) for 30 min at room temperature, thenextracted with methylene chloride and treated with triphenylphosphine inmethanol to reduce HPETEs to HETEs.

[0041]FIG. 7A: The products were analyzed by normal phase HPLC using anAlltech 5μ silica column (25×0.46 cm) and a solvent ofhexane/isopropanol/glacial acetic acid (100:2:0.1, by volume) at a flowrate of 1.1 ml/min. The column effluent was monitored using aHewlett-Packard 1040A diode array detector with an on-line PackardFlo-one radioactive detector.

[0042]FIG. 7B: The chirality of the 8-HETE product was analyzed as themethyl ester derivative using a Chiralcel OD column (25×0.46 cm) and asolvent of hexane/isopropanol (100:2, by volume) at a flow rate of 1.1ml/min.

[0043]FIG. 7C shows Western analyses of 8S-lipoxygenase expressed inHela cells, HEK cells and the enzyme from mouse skin. Lane 1: 15-Lox-1expressed in HEK cells (antibody does not recognize this protein). Lane2: 15-Lox-2, HEK cells. Lane 3: 8S-lipoxygenase in HEK cells. Lane 4:PMA-treated mouse skin. Lane 5: 8S-lipoxygenase in Hela cells. Lane 6:15-Lox-2 in Hela cells. All lanes were loaded with 5 μg protein, exceptlane 4 had only 2.5 μg protein.

[0044]FIG. 8 shows linoleic acid metabolism by 8S-lipoxygenase expressedin vaccinia virus-infected HeLa cells. Metabolism studies with[¹⁴C]linoleic acid (100 μM) used the same incubation and analysisconditions as described in the legend to FIG. 7.

[0045]FIG. 8A: Normal phase-HPLC of the products, analyzed afterreduction with triphenylphosphine.

[0046]FIG. 8B: Chiral HPLC analysis of the 9-HODE methyl ester.

[0047]FIG. 9 shows the effect of phorbol ester on 8S-lipoxygenaseexpression in mouse skin.

[0048]FIG. 9A: Normal-phase HPLC analysis of 8S-lipoxygenase activity inhomogenates of back skin of 7-8-day-old black Swiss pups following 24 hrtreatment with vehicle (acetone) or phorbol ester (50 nmol).

[0049]FIG. 9B: Northern analysis.

[0050]FIG. 9C: Western analysis.

[0051]FIG. 10 shows multiple tissue Northern analysis of mouse8S-lipoxygenase. A mouse tissue blot of mRNA (Clontech) was probed witha 618 bp EcoRV-BamHI fragment of 8S-lipoxygenase CDNA.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The lipoxygenase metabolism of arachidonic acid occurs inspecific blood cell types and epithelial tissues, and is activated ininflammation and tissue injury. In the course of studying lipoxygenaseexpression in human skin, a previously unrecognized enzyme was detectedand characterized that at least partly accounts for the 15S-lipoxygenasemetabolism of arachidonic acid in certain epithelial tissues. The cDNAwas cloned from human hair roots, and expression of the mRNA wasdetected also in prostate, lung, and cornea; an additional sixteen humantissues, including peripheral blood leukocytes, were negative for theMRNA. The CDNA encodes a protein of 676 amino acids with a calculatedmolecular weight of about 76 kD. The amino acid sequence hasapproximately 40% identity to the known human 5S-, 12S- and15S-lipoxygenases.

[0053] When expressed in human embryonic kidney (HEK) 293 cells, the newenzyme converts arachidonic acid exclusively to15S-hydroperoxyeicosatetraenoic acid, while linoleic acid is less wellmetabolized. These features contrast with the previously reported15S-lipoxygenase which oxygenates arachidonic acid mainly at C-15, butalso partly at C-12, and for which linoleic acid is an excellentsubstrate. The different catalytic activities and tissue distributionsuggest a distinct function for the new enzyme compared to thepreviously reported human 15S-lipoxygenase.

[0054] It is known that human hair roots metabolize arachidonic acid(Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297),and that in addition to a relatively prominent synthesis of 12S-HETE and15S-HETE, formation of minor amounts of 12R-HETE is detectable. (Baer etal. (1993) J. Lipid Research 34:1505-1514). Therefore freshly pluckedhuman hair follicles were as a source of RNA for the RT-PCR experimentsdescribed in Example 1. As described in Example 1, these experiments ledto the detection of a new lipoxygenase, a 15S-lipoxygenase (referred toherein as “15-Lox-2”) with a distinctive distribution in tissues.

Definitions and Techniques Affecting Gene Products and Genes

[0055] The present invention concerns DNA segments, isolatable fromvertebrate tissue, and preferably mammalian tissue, which are free fromgenomic DNA and which are capable of conferring arachidonic acidmetabolism activity in a recombinant host cell when incorporated intothe recombinant host cell. As used herein, the term “mammalian tissue”refers to, among others, normal mammalian epithelial tissues, asexemplified by, but not limited to, human embryonic kidney (HEK) 293cell lines. DNA segments capable of conferring arachidonic acidmetabolism activity may encode complete lipoxygenase gene products,cleavage products and biologically actively functional domains thereof.

[0056] The terms “lipoxygenase gene product”, “lipoxygenase”, “Lox”,“15-Lox-2 gene product”, “115-Lox-2”, “8-Lox gene product” and “8-Lox”as used in the specification and in the claims refer to proteins havingamino acid sequences which are substantially identical to the respectivenative lipoxygenase amino acid sequences and which are biologicallyactive in that they are capable of reacting with arachidonic acid or arecapable of cross-reacting with an anti-Lox antibody raised against alipoxygenase, such as 15-Lox-2 or 8-Lox. Such sequences are disclosedherein. The terms “lipoxygenase gene product”, “lipoxygenase”, “Lox”,“15-Lox-2 gene product”, “15-Lox-2”,“8-Lox gene product” and “8-Lox”also include analogs of lipoxygenase molecules which exhibit at leastsome biological activity in common with native lipoxygenase, 15-Lox-2,or 8-Lox. Furthermore, those skilled in the art of mutagenesis willappreciate that other analogs, as yet undisclosed or undiscovered, maybe used to construct lipoxygenase analogs. There is no need for a“lipoxygenase” or “Lox”, or a “15-Lox-2” or “8-Lox” to comprise all, orsubstantially all, of the amino acid sequence of the native lipoxygenasegenes. Shorter or longer sequences are anticipated to be of use in theinvention.

[0057] The terms “lipoxygenase gene”, “15-lox-2 gene” and “8-Lox gene”refer to any DNA sequence that is substantially identical to a DNAsequence encoding a lipoxygenase, 15-Lox-2 or 8-Lox as defined above.The terms also refer to RNA, or antisense sequences, compatible withsuch DNA sequences. A “lipoxygenase gene”, a “15-lox-2 gene” or a “8-Loxgene” may also comprise any combination of associated control sequences.

[0058] The term “substantially identical”, when used to define either alipoxygenase, a 15-lox-2 or a 8-Lox amino acid sequence, or alipoxygenase, a 15-lox-2 or a 8-Lox nucleic acid sequence, means that aparticular sequence, for example, a mutant sequence, varies from thesequence of a natural lipoxygenase, 15-lox-2 or 8-Lox by one or moredeletions, substitutions, or additions, the net effect of which is toretain at least some of biological activity of the lipoxygenase, the15-lox-2 or the 8-Lox protein. Alternatively, DNA analog sequences are“substantially identical” to specific DNA sequences disclosed herein if:(a) the DNA analog sequence is derived from coding regions of thenatural lipoxygenase, 15-lox-2 or 8-Lox gene; or (b) the DNA analogsequence is capable of hybridization of DNA sequences of (a) undermoderately stringent conditions and which encode biologically activelipoxygenase, 15-lox-2 or 8-Lox gene; or (c) the DNA sequences aredegenerative as a result of the genetic code to the DNA analog sequencesdefined in (a) and/or (b). Substantially identical analog proteins willbe greater than about 60% identical to the corresponding sequence of thenative protein. Sequences having lesser degrees of similarity butcomparable biological activity are considered to be equivalents. Indetermining nucleic acid sequences, all subject nucleic acid sequencescapable of encoding substantially similar amino acid sequences areconsidered to be substantially similar to a reference nucleic acidsequence, regardless of differences in codon sequences.

Percent Similarity

[0059] Percent similarity may be determined, for example, by comparingsequence information using the GAP computer program, available from theUniversity of Wisconsin Geneticist Computer Group. The GAP programutilizes the alignment method of Needleman et al. 1970, as revised bySmith et al. 1981. Briefly, the GAP program defines similarity as thenumber of aligned symbols (i.e. nucleotides or amino acids) which aresimilar, divided by the total number of symbols in the shorter of thetwo sequences. The preferred default parameters for the GAP programinclude: (1) a unitary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) of nucleotides and the weightedcomparison matrix of Gribskov et al., 1986, as described by Schwartz etal., 1979; (2) a penalty of 3.0 for each gap and an additional 0.01penalty for each symbol and each gap; and (3) no penalty for end gaps.

[0060] The term “homology” describes a mathematically based comparisonof sequence similarities which is used to identify genes or proteinswith similar functions or motifs. Accordingly, the term “homology” issynonymous with the term “similarity” and “percent similarity” asdefined above. Thus, the phrases “substantial homology” or “substantialsimilarity” have similar meanings.

Nucleic Acid Sequences

[0061] In certain embodiments, the invention concerns the use oflipoxygenase genes and gene products, such as the 15-lox-2 and 8-Loxgene products, that include within their respective sequences a sequencewhich is essentially that of a lipoxygenase, 15-lox-2 or 8-Lox gene, orthe corresponding proteins. The term “a sequence essentially as that oflipoxygenase, 15-lox-2 or 8-Lox gene or gene product”, means that thesequence substantially corresponds to a portion of a lipoxygenase,15-lox-2 or 8-Lox gene or gene product and has relatively few bases oramino acids (whether DNA or protein) which are not identical to those ofa lipoxygenase, 15-lox-2 or 8-Lox gene or gene product, (or abiologically functional equivalent of, when referring to proteins). Theterm “biologically functional equivalent” is well understood in the artand is further defined in detail herein. Accordingly, sequences whichhave between about 70% and about 80%; or more preferably, between about81% and about 90%; or even more preferably, between about 91% and about99%; of amino acids which are identical or functionally equivalent tothe amino acids of a lipoxygenase, 15-lox-2 or 8-Lox gene or geneproduct, will be sequences which are “essentially the same”.

[0062] Lipoxygenase, 15-lox-2 and 8-Lox genes which have functionallyequivalent codons are also covered by the invention. The term“functionally equivalent codon” is used herein to refer to codons thatencode the same amino acid, such as the six codons for arginine orserine, and also to refer to codons that encode biologically equivalentamino acids (see Table 1). TABLE 1 Functionally Equivalent Codons. AminoAcids Condons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGUAspartic Acid Asp D GAC GAU Glumatic acid Glu E GAA GAG PhenylalaninePhe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAUIsoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUGCUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU ProlinePro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGACGC CGG CGU Serine Ser S ACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACCACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr YUAC UAU

[0063] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences which may, for example, include variousnon-coding sequences flanking either of the 5′ or 3′ portions of thecoding region or may include various internal sequences, i.e., introns,which are known to occur within genes.

[0064] The present invention also encompasses the use of DNA segmentswhich are complementary, or essentially complementary, to the sequencesset forth in the specification. Nucleic acid sequences which are“complementary” are those which are base-pairing according to thestandard Watson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment in question under relativelystringent conditions such as those described herein.

[0065] Nucleic acid hybridization will be affected by such conditions assalt concentration, temperature, or organic solvents, in addition to thebase composition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1,000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. (See, e.g., Wetmur & Davidson, 1968).

[0066] Probe sequences may also hybridize specifically to duplex DNAunder certain conditions to form triplex or other higher order DNAcomplexes. The preparation of such probes and suitable hybridizationconditions are well known in the art.

[0067] As used herein, the term “DNA segment” refers to a DNA moleculewhich has been isolated free of total genomic DNA of a particularspecies. Furthermore, a DNA segment encoding a lipoxygenase, 15-lox-2 or8-Lox gene product refers to a DNA segment which contains lipoxygenase,15-lox-2 or 8-Lox coding sequences, yet is isolated away from, orpurified free from, total genomic DNA of Homo sapiens. Included withinthe term “DNA segment” are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phages, viruses, and the like.

[0068] Similarly, a DNA segment comprising an isolated or purifiedlipoxygenase, 15-lox-2 or 8-Lox gene refers to a DNA segment includinglipoxygenase, 15-lox-2 or 8-Lox coding sequences isolated substantiallyaway from other naturally occurring genes or protein encoding sequences.In this respect, the term “gene” is used for simplicity to refer to afunctional protein, polypeptide or peptide encoding unit. As will beunderstood by those in the art, this functional term includes bothgenomic sequences and cDNA sequences. “Isolated substantially away fromother coding sequences” means that the gene of interest, in this case,the lipoxygenase, 15-lox-2 or 8-Lox gene, forms the significant part ofthe coding region of the DNA segment, and that the DNA segment does notcontain large portions of naturally-occurring coding DNA, such as largechromosomal fragments or other functional genes or cDNA coding regions.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude genes or coding regions later added to the segment bythe hand of man.

[0069] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode a 15-Lox-2 protein that includes within its amino acid sequencethe amino acid sequence of SEQ ID NO:2. In other particular embodiments,the invention concerns isolated DNA segments and recombinant vectorsincorporating DNA sequences which encode a protein that includes withinits amino acid sequence the amino acid sequence of the 15-Lox-2 proteincorresponding to human epithelial tissue.

[0070] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode a 8-Lox protein that includes within its amino acid sequence theamino acid sequence of SEQ ID NO:4. In other particular embodiments, theinvention concerns isolated DNA segments and recombinant vectorsincorporating DNA sequences which encode a protein that includes withinits amino acid sequence the amino acid sequence of the 8-Lox proteincorresponding to mouse epithelial tissue.

[0071] It will also be understood that this invention is not limited tothe particular nucleic acid and amino acid sequences of SEQ ID NOS:1, 2,3 and 4. Recombinant vectors and isolated DNA segments may thereforevariously include the 15-Lox-2 and 8-Lox encoding regions themselves,include coding regions bearing selected alterations or modifications inthe basic coding region, or include encoded larger polypeptides whichnevertheless include 15-Lox-2 or 8-Lox encoding regions or may encodebiologically functional equivalent proteins or peptides which havevariant amino acid sequences.

[0072] In certain embodiments, the invention concerns isolated DNAsegments and recombinant vectors which encode a protein or peptide thatincludes within its amino acid sequence an amino acid sequenceessentially as set forth in SEQ ID NO:2 or SEQ ID NO:4. Naturally, wherethe DNA segment or vector encodes a full length 15-Lox-2 or 8-Lox geneproduct, the most preferred sequences are those which are essentially asset forth in SEQ ID NO:1 and SEQ ID NO:3 and which encode a protein thatexhibits arachidonic acid reactivity in HEK 293 cells, as may bedetermined by HPLC analysis, as disclosed herein.

[0073] The term “a sequence essentially as set forth in SEQ ID NO:2”means that the sequence substantially corresponds to a portion of SEQ IDNO:2 and has relatively few amino acids which are not identical to, or abiologically functional equivalent of, the amino acids of SEQ ID NO:2.The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences,which have between about 70% and about 80%; or more preferably, betweenabout 81% and about 90%; or even more preferably, between about 91% andabout 99%; of amino acids which are identical or functionally equivalentto the amino acids of SEQ ID NO:2, will be sequences which are“essentially as set forth in SEQ ID NO:2”. The term “a sequenceessentially set forth in SEQ ID NO:4” has a similar meaning.

[0074] In particular embodiments, the invention concerns gene therapymethods that use isolated DNA segments and recombinant vectorsincorporating DNA sequences which encode a protein that includes withinits amino acid sequence an amino acid sequence in accordance with SEQ IDNO:2 or in accordance with SEQ ID NO:4, SEQ ID NO:2 and SEQ ID NO:4derived from epithelial tissue from Homo sapiens. In other particularembodiments, the invention concerns isolated DNA sequences andrecombinant DNA vectors incorporating DNA sequences which encode aprotein that includes within its amino acid sequence the amino acidsequence of the 15-Lox-2 protein from human epithelial tissue, or whichencode a protein that includes within its amino acid sequence the aminoacid sequence of the 8-Lox protein from mouse epithelial tissue.

[0075] In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1, or anucleic acid sequence essentially as set forth in SEQ ID NO:3. The term“essentially as set forth in SEQ ID NO:1” is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, respectively, and hasrelatively few codons which are not identical, or functionallyequivalent, to the codons of SEQ ID NO:1, respectively. Again, DNAsegments which encode gene products exhibiting arachidonic acidmetabolism activity of the 15-Lox-2 and 8-Lox gene products will be mostpreferred. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, and also to refer to codons that encodebiologically equivalent amino acids (see Table 1). The term “essentiallyas set forth in SEQ ID NO:3” has a similar meaning.

[0076] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, nucleic acid fragments may beprepared which include a short stretch complementary to SEQ ID NO:1 orSEQ ID NO:3, such as about 10 nucleotides, and which are up to 10,000 or5,000 base pairs in length, with segments of 3,000 being preferred incertain cases. DNA segments with total lengths of about 1,000, 500, 200,100 and about 50 base pairs in length are also contemplated to beuseful.

[0077] The DNA segments of the present invention encompass biologicallyfunctional equivalent 15-Lox-2 and 8-Lox proteins and peptides. Suchsequences may rise as a consequence of codon redundancy and functionalequivalency which are known to occur naturally within nucleic acidsequences and the proteins thus encoded. Alternatively, functionallyequivalent proteins or peptides may be created via the application ofrecombinant DNA technology, in which changes in the protein structuremay be engineered, based on considerations of the properties of theamino acids being exchanged. Changes designed by man may be introducedthrough the application of site-directed mutagenesis techniques, e.g.,to introduce improvements to the antigenicity of the protein or to test15-Lox-2 and 8-Lox mutants in order to examine arachidonic acidreactivity at the molecular level.

[0078] If desired, one may also prepare fusion proteins and peptides,e.g., where the 15-Lox-2 or 8-Lox coding regions are aligned within thesame expression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins which may be purified by affinity chromatography and enzymelabel coding regions, respectively).

[0079] Recombinant vectors form important further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment is positionedunder the control of a promoter. The promoter may be in the form of thepromoter which is naturally associated with the 15-Lox-2 or 8-Loxgene(s), e.g., in epithelial cells, as may be obtained by isolating the5′ non-coding sequences located upstream of the coding segment or exon,for example, using recombinant cloning and/or PCR technology, inconnection with the compositions disclosed herein.

[0080] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a 15-Lox-2 or 8-Lox gene in itsnatural environment. Such promoters may include promoters isolated frombacterial, viral, eukaryotic, or mammalian cells. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type chosen for expression. The use ofpromoter and cell type combinations for protein expression is generallyknown to those of skill in the art of molecular biology, for example,see Sambrook et al., 1989, specifically incorporated herein byreference. The promoters employed may be constitutive, or inducible, andcan be used under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins or peptides. Appropriatepromoter systems contemplated for use in high-level expression include,but are not limited to, the vaccina virus promoter, which is more fullydescribed below.

[0081] As mentioned above, in connection with expression embodiments toprepare recombinant 15-Lox-2 and 8-Lox proteins and peptides, it iscontemplated that longer DNA segments will most often be used, with DNAsegments encoding the entire 15-Lox-2 or 8-Lox protein, functionaldomains or cleavage products thereof, being most preferred. However, itwill be appreciated that the use of shorter DNA segments to direct theexpression of 15-Lox-2 and 8-Lox peptides or epitopic core regions, suchas may be used to generate anti-15-Lox-2 or anti-8-Lox antibodies, alsofalls within the scope of the invention.

[0082] DNA segments which encode peptide antigens from about 15 to about50 amino acids in length, or more preferably, from about 15 to about 30amino acids in length are contemplated to be particularly useful. DNAsegments encoding peptides will generally have a minimum coding lengthin the order of about 45 to about 150, or to about 90 nucleotides. DNAsegments encoding full length proteins may have a minimum coding lengthon the order of about 5,600 nucleotides for a protein in accordance withSEQ ID NO:2 or a minimum coding length on the order of about 10,300nucleotides for a protein in accordance with SEQ ID NO:4.

[0083] Naturally, the present invention also encompasses DNA segmentswhich are complementary, or essentially complementary, to the sequenceset forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:3. Theterms “complementary” and “essentially complementary” are defined above.Excepting intronic or flanking regions, and allowing for the degeneracyof the genetic code, sequences which have between about 70% and about80%; or more preferably, between about 81% and about 90%; or even morepreferably, between about 91% and about 99%; of nucleotides which areidentical or functionally equivalent (i.e. encoding the same amino acid)of nucleotides of SEQ ID NO:1 or to the nucleotides of SEQ ID NO:3, willbe respectively sequences which are “essentially as set forth in SEQ IDNO:1” and will be sequences which are “essentially as set forth in SEQID NO:3”. Sequences which are essentially the same as those set forth inSEQ ID NO:1 or as those set forth in SEQ ID NO:3 may also befunctionally defined as sequences which are capable of hybridizing to anucleic acid segment containing the complement of SEQ ID NO:1 or to anucleic acid segment containing the complement of SEQ ID NO:3 underrelatively stringent conditions. Suitable relatively stringenthybridization conditions are described herein and will be well known tothose of skill in the art.

Biologically Functional Equivalents

[0084] As mentioned above, modification and changes may be made in thestructure of the lipoxygenase proteins and peptides, including 15-Lox-2and 8-Lox, described herein and still obtain a molecule having like orotherwise desirable characteristics. For example, certain amino acidsmay be substituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, C-15 carbon or C-8 carbon of arachidonic acid. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence (or, of course, itsunderlying DNA coding sequence) and nevertheless obtain a protein withlike or even countervailing properties (e.g., antagonistic v.agonistic). It is thus contemplated by the inventors that variouschanges may be made in the sequence of the lipoxygenase proteins andpeptides, including 15-Lox-2 and 8-Lox, (or underlying DNA) withoutappreciable loss of their biological utility or activity.

[0085] It is also well understood by the skilled artisan that, inherentin the definition of a biologically functional equivalent protein orpeptide, is the concept that there is a limit to the number of changesthat may be made within a defined portion of the molecule and stillresult in a molecule with an acceptable level of equivalent biologicalactivity. Biologically functional equivalent peptides are thus definedherein as those peptides in which certain, not most or all, of the aminoacids may be substituted. Of course, a plurality of distinctproteins/peptides with different substitutions may easily be made andused in accordance with the invention.

[0086] It is also well understood that where certain residues are shownto be particularly important to the biological or structural propertiesof a protein or peptide, e.g., residues in active sites, such residuesmay not generally be exchanged. This is the case in the presentinvention, where it any changes, for example, in an iron ligand moietyof 15-Lox-2 that render the peptide incapable of metabolism ofarachidonic acid to 15S-Hydro(pero)xyeicosatetraenoic acid would resultin a loss of utility of the resulting peptide for the present invention.

[0087] Amino acid substitutions, such as those which might be employedin modifying the lipoxygenase proteins and peptides, including 15-Lox-2and 8-Lox, described herein, are generally based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

[0088] In making such changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); praline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

[0089] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within +2 is preferred, those which are within +1 areparticularly preferred, and those within +0.5 are even more particularlypreferred.

[0090] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein. It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein.

[0091] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4)

[0092] In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0093] While discussion has focused on functionally equivalentpolypeptides arising from amino acid changes, it will be appreciatedthat these changes may be effected by alteration of the encoding DNA;taking into consideration also that the genetic code is degenerate andthat two or more codons may code for the same amino acid.

Sequence Modification Techniques

[0094] Modifications to the lipoxygenase proteins and peptides,including 15-Lox-2 and 8-Lox, described herein may be carried out usingtechniques such as site directed mutagenesis. Site-specific mutagenesisis a technique useful in the preparation of individual peptides, orbiologically functional equivalent proteins or peptides, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

[0095] In general, the technique of site-specific mutagenesis is wellknown in the art as exemplified by publications (e.g., Adelman et al.,1983). As will be appreciated, the technique typically employs a phagevector which exists in both a single stranded and double stranded form.Typical vectors useful in site-directed mutagenesis include vectors suchas the M13 phage (Messing et al., 1981). These phage are readilycommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis which eliminates the step of transferringthe gene of interest from a plasmid to a phage.

[0096] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartthe two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes, for example, the 15-Lox-2 and the8-Lox gene. An oligonucleotide primer bearing the desired mutatedsequence is prepared, generally synthetically, for example by the methodof Crea et al. (1978). This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

[0097] The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful 15-Lox-2, 8-Lox or other arachidonic acidmetabolizing species and is not meant to be limiting as there are otherways in which sequence variants of these peptides may be obtained. Forexample, recombinant vectors encoding the desired genes may be treatedwith mutagenic agents to obtain sequence variants (see, e.g., a methoddescribed by Eichenlaub, 1979) for the mutagenesis of plasmid DNA usinghydroxylamine.

Other Structural Equivalents

[0098] In addition to the lipoxygenase peptidyl compounds describedherein, the inventors also contemplate that other sterically similarcompounds may be formulated to mimic the key portions of the peptidestructure. Such compounds may be used in the same manner as the peptidesof the invention and hence are also functional equivalents. Thegeneration of a structural functional equivalent may be achieved by thetechniques of modeling and chemical design known to those of skill inthe art. It will be understood that all such sterically similarconstructs fall within the scope of the present invention.

Introduction of Gene Products

[0099] Where the gene itself is employed to introduce the gene products,a convenient method of introduction will be through the use of arecombinant vector which incorporates the desired gene, together withits associated control sequences. The preparation of recombinant vectorsis well known to those of skill in the art and described in manyreferences, such as, for example, Sambrook et al. (1989), specificallyincorporated herein by reference.

[0100] In vectors, it is understood that the DNA coding sequences to beexpressed, in this case those encoding the lipoxygenase gene products,are positioned adjacent to and under the control of a promoter. It isunderstood in the art that to bring a coding sequence under the controlof such a promoter, one generally positions the 5′ end of thetranscription initiation site of the transcriptional reading frame ofthe gene product to be expressed between about 1 and about 50nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. One mayalso desire to incorporate into the transcriptional unit of the vectoran appropriate polyadenylation site (e.g., 5′-AATAAA-3′), if one was notcontained within the original inserted DNA. Typically, these poly Aaddition sites are placed about 30 to 2000 nucleotides “downstream” ofthe coding sequence at a position prior to transcription termination.

[0101] While use of the control sequences of the specific gene (i.e.,the 15-Lox-2 promoter for 15-Lox-2) will be preferred, there is noreason why other control sequences could not be employed, so long asthey are compatible with the genotype of the cell being treated. Thus,one may mention other useful promoters by way of example, including,e.g., an SV40 early promoter, a long terminal repeat promoter fromretrovirus, an actin promoter, a heat shock promoter, a metallothioneinpromoter, and the like.

[0102] As is known in the art, a promoter is a region of a DNA moleculetypically within about 100 nucleotide pairs in front of (upstream of)the point at which transcription begins (i.e., a transcription startsite). That region typically contains several types of DNA sequenceelements that are located in similar relative positions in differentgenes. As used herein, the term “promoter” includes what is referred toin the art as an upstream promoter region, a promoter region or apromoter of a generalized eukaryotic RNA Polymerase II transcriptionunit.

[0103] Another type of discrete transcription regulatory sequenceelement is an enhancer. An enhancer provides specificity of time,location and expression level for a particular encoding region (e.g.,gene). A major function of an enhancer is to increase the level oftranscription of a coding sequence in a cell that contains one or moretranscription factors that bind to that enhancer. Unlike a promoter, anenhancer can function when located at variable distances fromtranscription start sites so long as a promoter is present.

[0104] As used herein, the phrase “enhancer-promoter” means a compositeunit that contains both enhancer and promoter elements. Anenhancer-promoter is operatively linked to a coding sequence thatencodes at least one gene product. As used herein, the phrase“operatively linked” means that an enhancer-promoter is connected to acoding sequence in such a way that the transcription of that codingsequence is controlled and regulated by that enhancer-promoter. Meansfor operatively linking an enhancer-promoter to a coding sequence arewell known in the art. As is also well known in the art, the preciseorientation and location relative to a coding sequence whosetranscription is controlled, is dependent inter alia upon the specificnature of the enhancer-promoter. Thus, a TATA box minimal promoter istypically located from about 25 to about 30 base pairs upstream of atranscription initiation site and an upstream promoter element istypically located from about 100 to about 200 base pairs upstream of atranscription initiation site. In contrast, an enhancer can be locateddownstream from the initiation site and can be at a considerabledistance from that site.

[0105] An enhancer-promoter used in a vector construct of the presentinvention can be any enhancer-promoter that drives expression in a cellto be transfected. By employing an enhancer-promoter with well-knownproperties, the level and pattern of gene product expression can beoptimized.

[0106] For introduction of, for example, the 15-Lox-2 or 8-Lox genes, itis proposed that one will desire to preferably employ a vector constructthat will deliver the desired gene to the affected cells. This will, ofcourse, generally require that the construct be delivered to thetargeted cells, for example, epthelial cells. It is proposed that thismay be achieved most preferably by introduction of the desired genethrough the use of a viral vector to carry either the 15-Lox-2 sequenceor the 8-Lox sequence to efficiently infect the cells. These vectorswill preferably be an adenoviral, a retroviral, a vaccinia viral vectoror adeno-associated virus. These vectors are preferred because they havebeen successfully used to deliver desired sequences to cells and tend tohave a high infection efficiency.

[0107] Commonly used viral promoters for expression vectors are derivedfrom polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40).The early and late promoters of SV40 virus are particularly usefulbecause both are obtained easily from the virus as a fragment which alsocontains the SV40 viral origin of replication. Smaller or larger SV40fragments may also be used, provided there is included the approximately250 bp sequence extending from the Hind III site toward the Bg1 I sitelocated in the viral origin of replication. Further, it is alsopossible, and often desirable, to utilize promoter or control sequencesnormally associated with the desired gene sequence, provided suchcontrol sequences are compatible with the host cell systems.

[0108] The origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or maybe provided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

[0109] Where the 15-Lox-2 or 8-Lox genes themselves are employed it willbe most convenient to simply use the wild type 15-Lox-2 gene or 8-Loxgene directly. However, it is contemplated that certain regions ofeither the 15-Lox-2 gene or the 8-Lox gene may be employed exclusivelywithout employing the entire wild type 15-Lox-2 or 8-Lox gene. It isproposed that it will ultimately be preferable to employ the smallestregion needed to regulate the metabolism of arachidonic acid to15S-hydro(pero)xyeicosatetraenoic acid or to8S-hydro(pero)xyeicosatetraenoic acid so that one is not introducingunnecessary DNA into cells which receive either a 15-Lox-2 geneconstruct or an 8-Lox gene construct. Techniques well known to those ofskill in the art, such as the use of restriction enzymes, will allow forthe generation of small regions of the 15-Lox-2 or 8-Lox genes. Theability of these regions to regulate the metabolism of arachidonic acidto 15S-hydro(pero)xyeicosatetraenoic acid or to8S-hydro(pero)xyeicosatetraenoic acid can easily be determined by theassays reported in the Examples. In general, techniques for assessingmetabolism of arachidonic acid to 15S-Hydro(pero)xyeicosatetraenoic acidor to 8S-hydro(pero)xyeicosatetraenoic acid are well known in the art.

Generation of Antibodies

[0110] In still another embodiment, the present invention provides anantibody immunoreactive with a polypeptide of the present invention.Preferably, an antibody of the invention is a monoclonal antibody. Meansfor preparing and characterizing antibodies are well known in the art(See, e.g., Antibodies A Laboratory Manual, E. Howell and D. Lane, ColdSpring Harbor Laboratory, 1988).

[0111] Briefly, a polyclonal antibody is prepared by immunizing ananimal with an immunogen comprising a polypeptide or polynucleotide ofthe present invention, and collecting antisera from that immunizedanimal. A wide range of animal species can be used for the production ofantisera. Typically an animal used for production of anti-antisera is arabbit, a mouse, a rat, a hamster or a guinea pig. Because of therelatively large blood volume of rabbits, a rabbit is a preferred choicefor production of polyclonal antibodies.

[0112] As is well known in the art, a given polypeptide orpolynucleotide may vary in its immunogenicity. It is often necessarytherefore to couple the immunogen (e.g., a polypeptide orpolynucleotide) of the present invention) with a carrier. Exemplary andpreferred carriers are keyhole limpet hemocyanin (KLH) and bovine serumalbumin (BSA). Other albumins such as ovalbumin, mouse serum albumin orrabbit serum albumin can also be used as carriers.

[0113] Means for conjugating a polypeptide or a polynucleotide to acarrier protein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0114] As is also well known in the art, immunogencity to a particularimmunogen can be enhanced by the use of non-specific stimulators of theimmune response known as adjuvants. Exemplary and preferred adjuvantsinclude complete Freund's adjuvant, incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

[0115] The amount of immunogen used of the production of polyclonalantibodies varies inter alia, upon the nature of the immunogen as wellas the animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal. The production of polyclonal antibodiesis monitored by sampling blood of the immunized animal at various pointsfollowing immunization. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored.

[0116] In another aspect, the present invention contemplates a processof producing an antibody immunoreactive with a lipoxygenase polypeptide,such as 15-Lox-2 or 8-Lox, the process comprising the steps of (a)transfecting recombinant host cells with a polynucleotide that encodesthat polypeptide; (b) culturing the host cells under conditionssufficient for expression of the polypeptide; (c) recovering thepolypeptide; and (d) preparing antibodies to the polypeptide.Preferably, the lipoxygenase polypeptide is capable of metabolizingarachidonic acid. Even more preferably, the present invention providesantibodies prepared according to the process described above.

[0117] A monoclonal antibody of the present invention can be readilyprepared through use of well-known techniques such as those exemplifiedin U.S. Pat. No 4,196,265, herein incorporated by reference. Typically,a technique involves first immunizing a suitable animal with a selectedantigen (e.g., a polypeptide or polynucleotide of the present invention)in a manner sufficient to provide an immune response. Rodents such asmice and rats are preferred animals. Spleen cells from the immunizedanimal are then fused with cells of an immortal myeloma cell. Where theimmunized animal is a mouse, a preferred myeloma cell is a murine NS-1myeloma cell.

[0118] The fused spleen/myeloma cells are cultured in a selective mediumto select fused spleen/myeloma cells from the parental cells. Fusedcells are separated from the mixture of non-fused parental cells, forexample, by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media. Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, the mediais supplemented with hypoxanthine.

[0119] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants forreactivity with an antigen-polypeptides. The selected clones can then bepropagated indefinitely to provide the monoclonal antibody.

[0120] By way of specific example, to produce an antibody of the presentinvention, mice are injected intraperitoneally with between about 1-200mg of an antigen comprising a polypeptide of the present invention. Blymphocyte cells are stimulated to grow by injecting the antigen inassociation with an adjuvant such as complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis). At some time (e.g., at least two weeks)after the first injection, mice are boosted by injection with a seconddose of the antigen mixed with incomplete Freund's adjuvant.

[0121] A few weeks after the second injection, mice are tail bled andthe sera titered by immunoprecipitation against radiolabeled antigen.Preferably, the process of boosting and titering is repeated until asuitable titer is achieved. The spleen of the mouse with the highesttiter is removed and the spleen lymphocytes are obtained by homogenizingthe spleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0122] Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

[0123] Myeloma cells are combined under conditions appropriate to fosterfusion with the normal antibody-producing cells from the spleen of themouse or rat injected with the antigen/polypeptide of the presentinvention. Fusion conditions include, for example, the presence ofpolyethylene glycol. The resulting fused cells are hybridoma cells. Likemyeloma cells, hybridoma cells grow indefinitely in culture.

[0124] Hybridoma ceils are separated from unfused myeloma cells byculturing in a selection medium such as HAT media (hypoxanthine,aminopterin, thymidine). Unfused myeloma cells lack the enzymesnecessary to synthesize nucleotides from the salvage pathway becausethey are killed in the presence of aminopterin, methotrexate, orazaserine. Unfused lymphocytes also do not continue to grow in tissueculture. Thus, only cells that have successfully fused (hybridoma cells)can grow in the selection media.

[0125] Each of the surviving hybridoma cells produces a single antibody.These cells are then screened for the production of the specificantibody immunoreactive with an antigen/polypeptide of the presentinvention. Single cell hybridomas are isolated by limiting dilutions ofthe hybridomas. The hybridomas are serially diluted many times and,after the dilutions are allowed to grow, the supernatant is tested forthe presence of the monoclonal antibody. The clones producing thatantibody are then cultured in large amounts to produce an antibody ofthe present invention in convenient quantity.

[0126] By use of a monoclonal antibody of the present invention,specific polypeptides and polynucleotide of the invention can berecognized as antigens, and thus identified. Once identified, thosepolypeptides and polynucleotide can be isolated and purified bytechniques such as antibody-affinity chromatography. Inantibody-affinity chromatography, a monoclonal antibody is bound to asolid substrate and exposed to a solution containing the desiredantigen. The antigen is removed from the solution through animmunospecific reaction with the bound antibody. The polypeptide orpolynucleotide is then easily removed from the substrate and purified.

Detecting a Polynucleotide or a Polypeptide of the Present Invention

[0127] Alternatively, the present invention provides a process ofdetecting a polypeptide of the present invention, wherein the processcomprises immunoreacting the polypeptides with antibodies preparedaccording to the process described above to form antibody-polypeptideconjugates, and detecting the conjugates.

[0128] In yet another embodiment, the present invention contemplates aprocess of detecting messenger RNA transcripts that encode a polypeptideof the present invention, wherein the process comprises hybridizing themessenger RNA transcripts with polynucleotide sequences that encode thepolypeptide to form duplexes; and detecting the duplex. Alternatively,the present invention provides a process of detecting DNA molecules thatencode a polypeptide of the present invention, wherein the processcomprises hybridizing DNA molecules with a polynucleotide that encodesthat polypeptide to form duplexes; and detecting the duplexes.

Screening Assays

[0129] In yet another aspect, the present invention contemplates aprocess of screening substances for their ability to affect arachidonicacid metabolism comprising the steps of providing a cell that contains afunctional polypeptide of the present invention and testing the abilityof selected substances to affect arachidonic acid metabolism in thatcell.

[0130] Utilizing the methods and compositions of the present invention,screening assays for the testing of candidate substances can be derived.A candidate substance is a substance which potentially can promote orinhibit arachidonic acid metabolism, by binding or other intramolecularinteraction, with a lipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox,that metabolizes arachidonic acid.

[0131] A screening assay of the present invention generally involvesdetermining the ability of a candidate substance to affect metabolism ofarachidonic acid in a target cell, such as the screening of candidatesubstances to identify those that inhibit or promote metabolism ofarachidonic acid. Target cells can be either naturally occurring cellsknown to contain a polypeptide of the present invention or transformedcell produced in accordance with a process of transformation set forthhereinbefore.

[0132] As is well known in the art, a screening assay provides a cellunder conditions suitable for testing arachidonic acid metabolism. Theseconditions include but are not limited to pH, temperature, tonicity, thepresence of relevant factors involved in arachidonic acid metabolism(e.g., metal ions such as for example Ca⁺⁺, growth factor, interleukins,or colony stimulating factors), and relevant modifications to thepolypeptide such as glycosylation or prenylation. It is contemplatedthat a polypeptide of the present invention can be expressed andutilized in a prokaryotic or eukaryotic cell. The host cell can also befractionated into sub-cellular fractions where the receptor can befound. For example, cells expressing the polypeptide can be fractionatedinto the nuclei, the endoplasmic reticulum, vesicles, or the membranesurfaces of the cell.

[0133] pH is preferably from about a value of 6.0 to a value of about8.0, more preferably from about a value of about 6.8 to a value of about7.8 and, most preferably about 7.4. In a preferred embodiment,temperature is from about 20° C. to about 50° C., more preferably fromabout 30° C. to about 40° C. and, even more preferably about 37° C.Osmolality is preferably from about 5 milliosmols per liter (mosm/L) toabout 400 mosm/l and, more preferably from about 200 milliosmols perliter to about 400 mosm/l and, even more preferably from about 290mosm/L to about 310 mosm/L. The presence of factors can be required forthe proper testing of arachidonic acid metabolism in specific cells.Such factors include, for example, the presence and absence (withdrawal)of growth factor, interleukins, or colony stimulating factors.

[0134] In one embodiment, a screening assay is designed to be capable ofdiscriminating candidate substances having selective ability to interactwith one or more of the polypeptides of the present invention but whichpolypeptides are without a substantially overlapping activity withanother of those polypeptides identified herein.

Screening Assays for a Polypeptide of the Present Invention

[0135] The present invention provides a process of screening abiological sample for the presence of a lipoxygenase polypeptide, suchas 15-Lox-2 or 8-Lox. Preferably, the lipoxygenase polypeptide reactswith arachidonic acid. A biological sample to be screened can be abiological fluid such as extracellular or intracellular fluid or a cellor tissue extract or homogenate. A biological sample can also be anisolated cell (e.g., in culture) or a collection of cells such as in atissue sample or histology sample. A tissue sample can be suspended in aliquid medium or fixed onto a solid support such as a microscope slide.

[0136] In accordance with a screening assay process, a biological sampleis exposed to an antibody immunoreactive with the polypeptide whosepresence is being assayed. Typically, exposure is accomplished byforming an admixture in a liquid medium that contains both the antibodyand the candidate polypeptide. Either the antibody or the sample withthe polypeptide can be affixed to a solid support (e.g., a column or amicrotiter plate).

[0137] The biological sample is exposed to the antibody under biologicalreaction conditions and for a period of time sufficient forantibody-polypeptide conjugate formation. Biological reaction conditionsinclude ionic composition and concentration, temperature, pH and thelike.

[0138] Ionic composition and concentration can range from that ofdistilled water to a 2 molal solution of NaCl. Preferably, osmolality isfrom about 100 mosmols/l to about 400 mosmols/l and, more preferablyfrom about 200 mosmols/l to about 300 mosmols/l. Temperature preferablyis from about 4° C. to about 100° C., more preferably from about 15° C.to about 50° C. and, even more preferably from about 25° C. to about 40°C. pH is preferably from about a value of 4.0 to a value of about 9.0,more preferably from about a value of 6.5 to a value of about 8.5 and,even more preferably from about a value of 7.0 to a value of about 7.5.The only limit on biological reaction conditions is that the conditionsselected allow for antibody-polypeptide conjugate formation and that theconditions do not adversely affect either the antibody or thepolypeptide.

[0139] Exposure time will vary inter alia with the biological conditionsused, the concentration of antibody and polypeptide and the nature ofthe sample (e.g., fluid or tissue sample). Means for determiningexposure time are well known to one of ordinary skill in the art.Typically, where the sample is fluid and the concentration ofpolypeptide in that sample is about 10⁻¹⁰M, exposure time is from about10 minutes to about 200 minutes.

[0140] The presence of polypeptide in the sample is detected bydetecting the formation and presence of antibody-polypeptide conjugates.Means for detecting such antibody-antigen (e.g., receptor polypeptide)conjugates or complexes are well known in the art and include suchprocedures as centrifugation, affinity chromatography and the like,binding of a secondary antibody to the antibody-candidate receptorcomplex.

[0141] In one embodiment, detection is accomplished by detecting anindicator affixed to the antibody. Exemplary and well known suchindicators include radioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), a secondantibody or an enzyme such as horse radish peroxidase. Means foraffixing indicators to antibodies are well known in the art. Commercialkits are available.

Screening Assay for Anti-polypeptide Antibody

[0142] In another aspect, the present invention provides a process ofscreening a biological sample for the presence of antibodiesimmunoreactive with a lipoxygenase polypeptide, such as 15-Lox-2 or8-Lox. Preferably the lipoxygenase polypeptide reacts with arachidonicacid. In accordance with such a process, a biological sample is exposedto a lipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox, underbiological conditions and for a period of time sufficient forantibody-polypeptide conjugate formation and the formed conjugates aredetected.

Screening Assay for Polynucleotide That Encodes a LipoxygenasePolypeptide, such as 15-Lox-2 or 8-Lox

[0143] A DNA molecule and, particularly a probe molecule, can be usedfor hybridizing as an oligonucleotide probe to a DNA source suspected ofencoding a lipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox.Preferably the lipoxygenase polypeptide reacts with arachidonic acid.The probing is usually accomplished by hybridizing the oligonucleotideto a DNA source suspected of possessing a lipoxygenase gene. In somecases, the probes constitute only a single probe, and in others, theprobes constitute a collection of probes based on a certain amino acidsequence or sequences of the polypeptide and account in their diversityfor the redundancy inherent in the genetic code.

[0144] A suitable source of DNA for probing in this manner is capable ofexpressing a polypeptide of the present invention and can be a genomiclibrary of a cell line of interest. Alternatively, a source of DNA caninclude total DNA from the cell line of interest. Once the hybridizationprocess of the invention has identified a candidate DNA segment, oneconfirms that a positive clone has been obtained by furtherhybridization, restriction enzyme mapping, sequencing and/or expressionand testing.

[0145] Alternatively, such DNA molecules can be used in a number oftechniques including their use as: (1) diagnostic tools to detect normaland abnormal DNA sequences in DNA derived from patient's cells; (2)means for detecting and isolating other members of the polypeptidefamily and related polypeptides from a DNA library potentiallycontaining such sequences; (3) primers for hybridizing to relatedsequences for the purpose of amplifying those sequences; (4) primers foraltering native lipoxygenase DNA sequences; as well as other techniqueswhich rely on the similarity of the DNA sequences to those of the DNAsegments herein disclosed.

[0146] As set forth above, in certain aspects, DNA sequence informationprovided by the invention allows for the preparation of relatively shortDNA (or RNA) sequences (e.g., probes) that specifically hybridize toencoding sequences of a selected lipoxygenase gene. In these aspects,nucleic acid probes of an appropriate length are prepared based on aconsideration of the encoding sequence for a polypeptide of thisinvention. The ability of such nucleic acid probes to specificallyhybridize to other encoding sequences lend them particular utility in avariety of embodiments. Most importantly, the probes can be used in avariety of assays for detecting the presence of complementary sequencesin a given sample. However, uses are envisioned, including the use ofthe sequence information for the preparation of mutant species primers,or primers for use in preparing other genetic constructions.

[0147] To provide certain of the advantages in accordance with theinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes probe sequences that are complementary to atleast a 14 to 40 or so long nucleotide stretch of a nucleic acidsequence of the present invention, such as that shown in SEQ ID NO:1. Asize of at least 14 nucleotides in length helps to ensure that thefragment is of sufficient length to form a duplex molecule that is bothstable and selective. Molecules having complementary sequences overstretches greater than 14 bases in length are generally preferred,though, to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 14 to 20 nucleotides, or even longerwhere desired. Such fragments can be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR technology of U.S.Pat. No. 4,683,202, herein incorporated by reference, or by introducingselected sequences into recombinant vectors for recombinant production.

[0148] Accordingly, a nucleotide sequence of the present invention canbe used for its ability to selectively form duplex molecules withcomplementary stretches of the gene. Depending on the applicationenvisioned, one employs varying conditions of hybridization to achievevarying degrees of selectivity of the probe toward the target sequence.For applications requiring a high degree of selectivity, one typicallyemploys relatively stringent conditions to form the hybrids. Forexample, one selects relatively low salt and/or high temperatureconditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C. to 70° C. Such conditions are particularly selective, and toleratelittle, if any, mismatch between the probe and the template or targetstrand.

[0149] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate polypeptide codingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions are typically needed to allowformation of the heteroduplex. Under such circumstances, one employsconditions such as 0.15M-0.9M salt, at temperatures ranging from 20° C.to 55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0150] In certain embodiments, it is advantageous to employ a nucleicacid sequence of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of giving a detectable signal. Inpreferred embodiments, one likely employs an enzyme tag such a urease,alkaline phosphatase or peroxidase, instead of radioactive or otherenvironmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known which can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

[0151] In general, it is envisioned that the hybridization probesdescribed herein are useful both as reagents in solution hybridizationas well as in embodiments employing a solid phase. In embodimentsinvolving a solid phase, the sample containing test DNA (or RNA) isadsorbed or otherwise affixed to a selected matrix or surface. Thisfixed, single-stranded nucleic acid is then subjected to specifichybridization with selected probes under desired conditions. Theselected conditions depend inter alia on the particular circumstancesbased on the particular criteria required (depending, for example, onthe G+ C contents, type of target nucleic acid, source of nucleic acid,size of hybridization probe, etc.). Following washing of the hybridizedsurface so as to remove nonspecifically bound probe molecules, specifichybridization is detected, or even quantified, by means of the label.

Assay Kits

[0152] In another aspect, the present invention contemplates diagnosticassay kits for detecting the presence of a polypeptide of the presentinvention in biological samples, where the kits comprise a firstcontainer containing a first antibody capable of immunoreacting with thepolypeptide, with the first antibody present in an amount sufficient toperform at least one assay. Preferably, the assay kits of the inventionfurther comprise a second container containing a second antibody thatimmunoreacts with the first antibody. More preferably, the antibodiesused in the assay kits of the present invention are monoclonalantibodies. Even more preferably, the first antibody is affixed to asolid support. More preferably still, the first and second antibodiescomprise an indicator, and, preferably, the indicator is a radioactivelabel or an enzyme.

[0153] The present invention also contemplates a diagnostic kit forscreening agents. Such a kit can contain a polypeptide of the presentinvention. The kit can contain reagents for detecting an interactionbetween an agent and a receptor of the present invention. The providedreagent can be radiolabelled. The kit can contain a known radiolabelledagent capable of binding or interacting with a receptor of the presentinvention.

[0154] In an alternative aspect, the present invention providesdiagnostic assay kits for detecting the presence, in biological samples,of a polynucleotide that encodes a polypeptide of the present invention,the kits comprising a first container that contains a secondpolynucleotide identical or complementary to a segment of at least 10contiguous nucleotide bases of, as a preferred example, SEQ ID NO:1 orSEQ ID NO:3.

[0155] In another embodiment, the present invention contemplatesdiagnostic assay kits for detecting the presence, in a biologicalsample, of antibodies immunoreactive with a polypeptide of the presentinvention, the kits comprising a first container containing alipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox, that immunoreactswith the antibodies, with the polypeptide present in an amountsufficient to perform at least one assay. Preferably, the lipoxygenasepolypeptide metabolizes arachidonic acid. The reagents of the kit can beprovided as a liquid solution, attached to a solid support or as a driedpowder. Preferably, when the reagent is provided in a liquid solution,the liquid solution is an aqueous solution. Preferably, when the reagentprovided is attached to a solid support, the solid support can bechromatograph media or a microscope slide. When the reagent provided isa dry powder, the powder can be reconstituted by the addition of asuitable solvent. The solvent can be provided.

[0156] The following examples have been included to illustrate preferredmodes of the invention. Certain aspects of the following examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These examples are exemplified through the use of standard laboratorypractices of the inventors. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

EXAMPLE 1 ISOLATION OF A SECOND 15S-LIPOXYGENASE (15-LOX-2)

[0157] Preparation of Total RNA, and cDNA Synthesis

[0158] For each RNA preparation, about 50 human scalp hairs were pluckedindividually from a volunteer. About 30 hair roots, mainly from anagenfollicles (Baden et al. (1979) J. Amer. Acad. Dermatol. 1:121-122), werecut off and dropped into 1 ml of guanidinium thiocyanate solution, thelysis buffer from the RNeasy RNA extraction kit (Qiagen). After a briefsonication using an ultrasonic probe (2 sec, twice), total RNA wasextracted according to the manufacturer's instructions. Approximately5-10 μg of total RNA was recovered in 50 μl of water. In someexperiments, RNA was prepared from psoriatic scales using essentiallythe same procedure. Thirty microliter aliquots of RNA were used in 50 μlreactions for first strand cDNA synthesis using either anoligo-dT-adaptor primer, random hexamer primers, or the Marathon RACEprocedure (Clontech) as described previously (Brash et al. (1996) J.Biol. Chem. 271, 20549-20557). One microliter aliquots of cDNA were useddirectly in PCR reactions.

[0159] PCR Experiments

[0160] The primers encoded conserved sequences in animal and plantlipoxygenases. Two upstream primers encoded the sequence WLLAK (SEQ IDNO:5) from the middle of the lipoxygenase primary structure. Thissequence forms the beginning of a long helix that crosses the center ofthe protein and includes two of the histidine iron ligands. The twoupstream primers differed only in using alternative codons for the 3′lysine, AAA or AAG, and were designated as WLLAK-(AAA) and WLLAK-(AAG):5′-GAC-GTC-TGG-YTi-YTi-GCi-AAA, (SEQ ID NO:6) or -AAG-3′ (SEQ ID NO:7)(where i encodes inosine). The human 5S-lipoxygenase and the blood cell15S-lipoxygenase are encoded as WLLAK-(AAA) (SEQ ID NO:6) (Matsumoto etal. (1988) Proc. Natl. Acad. Sci. USA 85; 26-30; Dixon et al. (1988)Proc. Natl. Acad. Sci. USA 85, 416-420; Sigal et al. (1988) Biochem.Biophys. Res. Comm. 157, 457-464), whereas the platelet 12-lipoxygenaseuses WLLAK-(AAG) (SEQ ID NO:7) (Table 2) (Funk et al. (1990) Proc. Natl.Acad. Sci. USA 87, 5638-5642; Izumi et al. (1990) Proc. Natl. Acad. Sci.USA 87, 7477-7481). (One of the three papers on the human platelet12-lipoxygenase reports a different sequence around this lysine,Yoshimoto et al. (1990) Biochem. Biophys. Res. Comm. 172, 1230-1235.)

[0161] For the first round PCR, each upstream primer was used inseparate reactions against a set of downstream primers encoding an aminoacid sequence that occurs seven amino acids downstream of the most 3′histidine ligand to the lipoxygenase iron on a second long helix. Thesequence GQLDW (SEQ ID NO:8) occurs in the human 12S- and15S-lipoxygenases beginning at amino acid position 546 and was encoded(with an additional three amino acids of consensus sequence on the 5′end) as 5′-CCA-AGT-GTA-CCA-RTC-NAG-YTG-NCC-3′ (SEQ ID NO:9). Thesequence GQYDW (SEQ ID NO:35) occurs in the equivalent position in thehuman 5S-lipoxygenase and this primer differed only in changing oneamino acid code from leucine to tyrosine(5′-CCA-AGT-GTA-CCA-RTC-RTA-YTG-NCC-3′) (SEQ ID NO:10).

[0162] The first round PCR reaction was primed with human hair folliclecDNA and in some experiments with cDNA prepared from psoriatic scales (1μl from a 50 μl reaction using 5 μg total RNA) per 50 μl PCR reaction,and using 10 mM Tris, pH 8.3, 50 mM KCl, 3 mM MgCl₂ with 0.2 mM of eachdNTP and 0.25 μl (1.25 units) AmpliTaq DNA polymerase (Perkin Elmer) ina Perkin Elmer 480 thermocycler. After addition of cDNA at 80° (hotstart), the PCR was programmed as follows: 940 for 2 min, 1 cycle; 50°for 1 min, 72° for 1 min, 94° for 1 min, 30 cycles; 72° for 10 min, 1cycle, and then the block temperature was held at 4° C.

[0163] For second round PCR, the upstream primer was either retained asbefore (WLLAK-(AAA) (SEQ ID NO:6) or WLLAK-(AAG) (SEQ ID NO:7)), orchanged to a nested upstream primer modified very slightly from thatused by Funk and colleagues (Funk et al. (1990) Proc. Natl. Acad. Sci.USA 87:5638-5642) for cloning of the human 12S-lipoxygenase and encodingthe sequence XVDWLLAKXWVR (SEQ ID NO:36):5′-TA-GTC-GAC-TGG-CTT-YTG-GCC-AAA-iiC-TGG-GTS-CG-3′ (where S (“strong”)encodes C or G) (SEQ ID NO:11). The downstream primer for all secondround reactions (nested PCR) encoded the sequence ELQXWWR (SEQ ID NO:26)and included a BamHI restriction site at the 5′ end:5′-G-CGG-ATC-CCT-CCA-CCA-GGN-YTG-SAG-YTC-3′ (SEQ ID NO:12). The secondround PCR reactions used 1 μl of 10-times dilute first round PCRproducts as cDNA and otherwise the conditions differed only in usingeither 55° or 58° as annealing temperature.

[0164] 3′ RACE and 5′ RACE

[0165] The 3′ sequence was obtained using established upstream sequencefor the new human lipoxygenase (first round:5′-GGT-ATC-TAC-TAC-CCA-AGT-GAT-GAG-3′ (SEQ ID NO:13); second round:5′-TAC-CCA-AGT-GAT-GAG-TCT-GTC-3′ (SEQ ID NO:14)) against a downstreamprimer based on the adaptor-linked oligo-dT primer used for cDNAsynthesis, as described previously (Brash et al. (1996) J. Biol. Chem.271:20549-20557). The 5′ RACE was accomplished using the Marathon cDNAAmplication Kit (Clontech) (Brash et al. (1996) J. Biol. Chem.271:20549-20557) using 4 μg of total RNA from beard hair follicles. Thegene-specific downstream primers were5′-GAA-GAC-CTC-AGG-CAG-CAG-ATG-TG-3′ (SEQ ID NO:15) and5′-TC-ATG-GAA-GGA-GAA-CTC-GGC-AT-3′ (SEQ ID NO:16). A full length clonewas obtained by PCR using primers purified by HPLC (Brash et al. (1996)J. Biol. Chem. 271:20549-20557) and using a proof-reading mixture ofTaq/Pwo DNA polymerases (Expand High Fidelity, Boehringer-Mannheim) asdescribed (Brash et al. (1996) J. Biol. Chem. 271:20549-20557). Theupstream primer encoded the N-terminus with a BamHI site added at the 5′end to facilitate subcloning: 5′AC-GGA-TCC-AGC-ATG-GCC-GAG-TTC-AGG-GTC-AG 3′ (SEQ ID NO:17), and thedownstream primer encoded the C-terminus of the protein with an added5′EcoRI site to facilitate subcloning: 5′CGG-AAT-TCA-TGT-CAT-CTG-GGC-CTG-TGT-TCC 3′ (SEQ ID NO:18). After a hotstart at 80° C., the reaction conditions were 94°, 2 min, 1 cycle; 58°for 30 sec, 72° for 1 min 30 sec, 96° 15 sec, 3 cycles; 68° for 2 min,96° 15 sec, 30 cycles; 72° 10 min, 1 cycle; hold at 4° C.

[0166] Northern Analysis

[0167] Two nylon membranes containing MRNA from human tissues (Clontech,Palo Alto, Calif.) were probed using a ³²P-labeled 1059 bp fragment ofthe new human lipoxygenase prepared from the plasmid by PCR (withprimers 5′-TG-CCT-CTC-GCC-ATC-CAG-CT-3′ (SEQ ID NO:19) and 5′TG-TTC-CCC-TGG-GAT-TTA-GAT-GGA-3′) (SEQ ID NO:20) and labeled byRediprime random priming (Amersham). After hybridization in ExpressHybsolution (Clontech) at 68° C. for 1 hr, the membranes were washedfinally in 0.1×SSC/0.1% SDS at 50° C. for 40 min and exposed to film.

[0168] Detection of the cDNA in Human Cornea

[0169] RNA was prepared using Tri Reagent (Molecular Research Center,Inc., Cincinnati, Ohio) from corneal epithelial cells scraped from eyebank corneas unsuitable for transplantation. The RNA samples weretreated with DNAse 1, then reverse transcribed to cDNA. PCR reactionswere run with human cornea CDNA as template, and also with rabbit corneaCDNA and buffer alone as negative controls. Additional negative controlsusing RNA without the reverse transcriptase step confirmed the absenceof DNA contaminants in the samples. Two pairs of primers were used:GGT-ATC-TAC-TAC-CCA-AGT-GAT-GAG (SEQ ID NO:21) with5′-TGGGATGTCATCTGGGCCTGT-3′ (SEQ ID NO:22) giving a 589 bp product (#1),and from the 3′ untranslated region (UTR), #2:5′-AACTCACCCCCACCACCATACACA-3′ (SEQ ID NO:23) with5′-TTCCCGCCTCCATCTCCCAAAGT-3′ (SEQ ID NO:24) giving a 351 bp product (#2). Both reactions were run using an annealing temperature of 65° in thePCR. Northern analysis of eye tissues used approximately 1 μg of polyA-selected RNA and the same hybridization protocol as given above.

[0170] DNA Sequencing

[0171] PCR products were subcloned into the pCR2.1 vector (Invitrogen)and sequenced using the Oncor Fidelity manual dideoxy chain terminationmethod or by automated sequencing on a ABI Prism 310 Genetic analyzerand fluorescence-tagged dye terminator cycle sequencing (Perkin Elmer).

[0172] Expression of cDNA, HPLC Analysis of Lipoxygenase Metabolism

[0173] The PCR products corresponding to the open reading frame of thecDNA were subcloned into the pCDNA3 vector (Invitrogen), or in someexperiments ligated directly into pCR3 (Invitrogen), and expressed bytransient transfection in human embryonic kidney (HEK) 293 cells asdescribed (Funk et al. (1996) J. Biol. Chem. 271, 23338-23344).Following incubation with substrate (100 uM [1-¹⁴C]arachidonic acid or[1-¹⁴C]linoleic acid) for 30 min at 37° C., products were extractedusing the Bligh and Dyer procedure (Bligh et al. (1959) Can. J. Biochem.Physiol. 37:911-917) and the extracts were analyzed by reversed-phaseHPLC, straight phase HPLC and chiral column analysis (Brash et al.(1990) Method. Enzymol. 187:187-192).

[0174] Results of PCR Experiments

[0175] As described more fully in Experimental Procedures and summarizedin Table 2, a PCR strategy was developed using sets of degenerateupstream and downstream primers that would resolve the known 5S-, 12S-,and 15S-lipoxygenases into separate tubes. The reactions were run undernon-stringent conditions to permit detection of related sequences. Aftertwo rounds of reactions (nested PCR, see Experimental Procedures),successful amplification was expected to give a PCR product ofapproximately 500 bp.

[0176] When the reactions were carried out using different human hairroot cDNAs as template, bands of ≈500 bp were evident in tubescorresponding to several of the original combinations of primer. Many ofthe bands were found to represent the known 12S- and 15S-lipoxygenasesequences. These two cDNAs were successfully resolved into separate PCRreactions by making use of their different codon usages for lysine 344at the 3′ end of the upstream primer (Table 2, and ExperimentalProcedures). Over 60 clones from the first two primer combinations inTable 2 were categorized as 12- or 15-lipoxygenase by sequencing and/orrestriction enzyme digest with ApaI and HindIII.

[0177] Particular attention was paid to the 500 bp product obtained fromthe fourth primer set in Table 2, as this combination of sequences isnot found in the three previously cloned human lipoxygenases. Of 41clones with the correct sized insert, 39 cut with ApaI as expected ofthe human 12S-lipoxygenase. These clones appeared to correspond to12S-lipoxygenase cDNA that had annealed to the slightly mismatchedprimers under the non-stringent conditions of PCR; a limited number weresequenced and all were identical to the human 12S-lipoxygenase. Two ofthe 41 positive clones did not cut with ApaI or HindIII, and sequencingindicated these clones represented a new lipoxygenase cDNA. The completecDNA sequence of this new lipoxygenase was extended by 3′ RACE and 5′RACE, and full length clones corresponding to the open reading framewere obtained by PCR. Two of the active clones (see below) were fullysequenced. The percent identity to the reported amino acid sequences ofthe 5S-, 12S- and 15S-lipoxygenases are approximately 44% to the5-lipoxygenase, and 38-39% to the 12- and 15-lipoxygenases. FIG. 1 showsthe deduced amino acid sequence (SEQ ID NO:1) in alignment with the15S-lipoxygenase of human blood cells (SEQ ID NO:25) (Sigal et al.(1988) Biochem. Biophys. Res. Comm. 157, 457-464).

[0178] Results of Expression Studies

[0179] Initially, five full length clones were expressed in HEK 293cells and the lipoxygenase activity evaluated by incubation with[¹⁴C]arachidonic acid followed by HPLC analysis. Three of the PCR clonesexpressed with equivalent activity. The active clones made a singleproduct, identified as 15-HETE (after reduction of the HPETE) on thebasis of its retention time on reversed-phase HPLC (FIG. 2A) andSP-HPLC, and its characteristic uv spectrum (Ingram et al. (1988) Lipids23:340-344); it was exclusively the 15S enantiomer as determined bychiral column analysis (FIG. 2B). The same product was formed followingexpression in Hela cells and Cos cells, and in these experiments anothertwenty clones, eight active, were evaluated. Addition of calcium (2 mM)or ATP (2 mM) to the incubation media had no significant effect onenzymatic activity.

[0180] Differences from the 15S-lipoxygenase of Blood Cells

[0181] Applicants looked carefully for any 12-HETE or other HETEby-products of the new 15S-lipoxygenase and unexpectedly, found none.This is in sharp contrast to the 15S-lipoxygenase of human blood cellsthat was analyzed in the same experiments; as reported before, the bloodcell 15S-lipoxygenase forms 10-20% 12S-HETE in addition to 15S-HETE(Bryant et al. (1982) J. Biol. Chem. 257:6050-6055).

[0182] A comparison of the metabolism of arachidonic acid and linoleicacid revealed a second significant difference between the two enzymes.Linoleic acid is an excellent substrate for the blood cell15S-lipoxygenase (Soberman et al. (1985) J. Biol. Chem. 260:4508-4515);in applicants' experiments it was metabolized more extensively thanarachidonic acid. Although the new 15-lipoxygenase did metabolizelinoleic acid, it was not as good a substrate. In two experiments,linoleic acid was 11% and 37% metabolized by the new enzyme, while therespective values for arachidonic acid were 30% and 83% conversion.

[0183] Expression in Other Tissues

[0184] Multiple tissue Northern blots showed fourteen tissues negativefor the new 15S-lipoxygenase mRNA (heart, brain, placenta, liver,skeletal muscle, kidney, pancreas, spleen, thymus, testis, ovary, smallintestine, colon, and peripheral blood leukocytes) and two distinctlypositive (FIG. 3). The positive tissues, lung and prostate, showed atranscript estimated as 2.5-3 kb, compatible with the established sizeof the cDNA (2.7 kb). Applicants also checked for the presence in cornea(originally because of a suspected connection to 12R-HETE synthesis). Asdetermined by RT-PCR, human cornea is positive for the new lipoxygenasemRNA (FIG. 4a), and Northern analysis confirmed the presence of the newlipoxygenase transcript (FIG. 4b).

[0185] The human lipoxygenases can be distinguished by their positionalspecificity, by other distinctive features of their catalytic activitiessuch as their ability to metabolize Cie fatty acid substrates, by theircellular distribution, and functionally, in their physiological roles.Funk, C. D. (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98. The new15S-lipoxygenase characterized herein has a distinctive substratespecificity, a unique tissue distribution, and a different physiologicalrole from the previously known human 15S-lipoxygenase.

[0186] The primary structure of the new enzyme has the features typicalof a lipoxygenase. It has about 40% amino acid sequence identity to theblood cell 15S-lipoxygenase and other reported mammalian lipoxygenases.The sequence contains the absolutely conserved iron-binding histidinesand the carboxy terminal isoleucine that also functions as an ironligand (Boyington et al. (1993) Science 260:1482-1486; Minor et al.(1996) Biochemistry 35:10687-10701). One difference from other membersof the lipoxygenase gene family is a change in the putative fifth ironligand, normally a histidine or asparagine (N693 in the soybean L1enzyme (Boyington et al. (1993) Science 260:1482-1486; Minor et al.(1996) Biochemistry 35:10687-10701), H544 in the human blood cell15S-lipoxygenase (Sigal et al. (1988) Biochem. Biophys. Res. Comm.157:457-464). In the new human lipoxygenase the equivalent residue ischanged to a serine (S558).

[0187] Catalytically the enzyme differs from the blood celliSS-lipoxygenase in two important respects: it oxygenates moreexclusively at the 15 carbon, and linoleic acid is a relatively poorsubstrate. These two features of the new 15S-lipoxygenase, the highpositional specificity and the preference for arachidonic acid, have aparallel among 12-lipoxygenases in the properties of the12S-lipoxygenase of platelets (Nugteren, D. H. (1975) Biochim. Bikophys.Acta 380:299-307;Hada et al. (1991) Biochim. Biophys. Acta 1083:89-93).Given this analogy in catalytic activities, it is likely that the newenzyme will be a comparatively poor metabolizer of esterified fattyacids, in contrast to the blood cell 15S-lipoxygenase (Schewe et al.(1975) FEBS Lett. 60:149-153; Murray et al. (1988) Arch. Biochem.Biophys. 265:514-523).

[0188] The four tissues in which the new enzyme is located, skin, lung,prostate and cornea, are all reported sites of 15-HETE synthesis. Inhuman skin, applicants have now established the occurrence of both typesof 15S-lipoxygenase. In lung, the 15-HETE synthesis has been ascribed tothe blood cell type of 15-lipoxygenase (Sigal et al. (1992) Am. J.Physiol. 262:L392-398) and this enzyme has been detected byimmunohistochemistry (Nadel et al. (1991) J. Clin. Invest. 87:1139-1145;Shannon et al. (1991) Am. J. Physiol. 261:L399-405; Shannon et al.(1993) Am. Rev. Respir. Dis. 147:1024-1028). But, clearly thepossibility that the new 15-lipoxygenase contributes to the synthesis incertain cell types should be re-examined. Applicants' finding of themRNA in prostate is compatible with the reports by Oliw and colleaguesof the occurrence of 15-lipoxygenase in prostasomes, components of semensecreted by the prostate gland. Oliw et al. (1989) Biochim. Biophys.Acta 1002:283-291; Oliw et al. (1993) J. Reprod. Fertil. 99, 195-199.Similarly, our detection of the cDNA in cornea is in accord withmetabolism studies by Oliw and coworkers; they established that humancornea synthesizes 15S-HETE from [¹⁴C]arachidonic acid. Liminga et al.(1994) Biochim. Biophys. Acta 1210:288-296. Additional studies usingimmunohistochemistry indicated expression of the blood cell type of15S-lipoxygenase in human cornea. Liminga et al. (1994) Exp. Eye Res.59, 313-321. In cornea, as in skin, it is likely that both types of15S-lipoxygenase are expressed.

[0189] It appears that this enzyme is a lipoxygenase specific forcertain epithelial tissues. Based on the Northern result on colon andsmall intestine, the enzyme is not expressed in all epithelia, but thetissues in which it is identified so far are epithelial or have asignificant epithelial component. As described in Example 2, the newhuman enzyme is related in primary structure to the phorbol esterinducible 8S-lipoxygenase of mouse skin. Thus, regulation of theexpression of the new human enzyme is a significant feature of itsinvolvement in the pathophysiology of skin and other tissues. TABLE 2Primers (first round PCR) to resolve human lipoxygenases Reference forUpstream Downstream Match to known known Primer Primer^(a) lipoxygenaselipoxygenase WLLAK-(AAA) GQLDW 15S-lipoxygenase Sigal et al. (SEQ IDNO:6) (SEQ ID NO:8) (1988) WLLAK-(AAG) GQLDW 12S-lipoxygenase Funk etal. (SEQ ID NO:7) (SEQ ID NO:8) (1990); Izumi et al. (1990) WLLAK-(AAA)GQYDW 5S-lipoxygenase Matsumoto et (SEQ ID NO:6) (SEQ ID NO:35) al.(1988); Dixon et al. (1988) WLLAK-(AAG) GQYDW NONE NONE (SEQ ID NO:7)(SEQ ID NO:35)

EXAMPLE 2 Molecular Cloning and Functional Expression of a PhorbolEster-inducible 8S-lipoxygenase (8-Lox) from Mouse Skin

[0190] As described above, in the course of studies on HETE synthesis inskin, a second type of 15S-lipoxygenase from human skin was cloned. Thisenzyme is different from the well known reticulocyte-type of15S-lipoxygenase in that it oxygenates arachidonic acid purely at C-15and linoleic acid is a relatively poor substrate. Continuing with theabbreviations adopted above, the reticulocyte-type of 15S-lipoxygenaseis referred to as 15-Lox-1 and the enzyme that is an aspect of thisinstant invention is referred to as 15-Lox-2 in this example.

[0191] It was not clear a priori what is the animal homologue of the newhuman lipoxygenase, 15-Lox-2. In searching for a potential murinehomologue a series of PCR reactions using mouse skin were performed.This led to the detection of a new mouse cDNA that is characterized inthis example.

[0192] One of the effects of topical application of phorbol ester tomouse skin is the induction of an 8S-lipoxygenase in association withthe inflammatory response. This example describes the molecular cloningand characterization of this enzyme. The cDNA was isolated by PCR frommouse epidermis and subsequently from a mouse epidermal cDNA library.The cDNA encodes a protein of 677 amino acids with a calculatedmolecular weight of 76 kDa. The amino acid sequence has 78% identity to15-Lox-2, and approximately 40% to other mammalian lipoxygenases. Whenexpressed in vaccinia virus-infected Hela cells, the mouse enzymeconverts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoicacid, while linoleic acid is converted to 9S-hydroperoxy-linoleic acidin lower efficiency.

[0193] Phorbol ester treatment of mouse skin is associated with stronginduction of 8S-lipoxygenase mRNA and protein. By Northern analysis,expression of 8S-lipoxygenase mRNA was also detected in brain.Immunohistochemical analysis of phorbol ester-treated mouse skin showedthe strongest reaction to 8S-lipoxygenase in the differentiatedepidermal layer, the stratum granulosum. The inducibility of this enzymeis likely a characteristic feature of the mouse 8S-lipoxygenase and itshuman 15S-lipoxygenase homologue.

[0194] Preparation of Mouse Epidermal Total RNA, and cTNa Synthesis

[0195] Phorbol ester (PMA, 10 nmol) dissolved in 50 ml of acetone wasapplied topically onto dorsal skin of 6-7-day-old mice. At 21-24 h afterPMA-treatment, the mice were euthanized, and epidermis was prepared fromthe frozen dorsal skin as previously described (Hughes et al. (1991)Biochim. Biophys. Acta 1081:347-354). The frozen epidermis was droppedinto guanidinium thiocyanate solution, the lysis buffer from the RNeasyRNA extraction kit (QIAGEN). After a brief sonication using anultrasonic probe (2 sec, twice), total RNA was extracted according tothe manufacturer's instructions. Approximately 50 mg of total RNA wasrecovered in 50 ml of water. Twenty microliter aliquots were used in 50ml reactions for first strand cDNA synthesis using an oligo-dT-adaptorprimer (Brash et al. (1996) J. Biol. Chem. 271:20949-20957). Onemicroliter aliquots of CDNA were used directly in PCR reactions.

[0196] PCR Cloning of Epidermal Lipoxygenase cDNA—Initial PCR Clone

[0197] Two upstream degenerate primers encoded the sequence DVWLLAK (SEQID NO:27). The two primers differed only in using alternative codons forthe 3′ lysine, AAA or AAG, and they are referred to as WLLAK-(AAA) (SEQID NO:6) and WLLAK-(AAG) (SEQ ID NO:7). For the first round PCRreaction, each upstream primer was used in separate reactions against aset of downstream primers encoding three amino acid sequences beginningGQ that occur seven amino acids downstream of the most 3′ histidineligand to the iron: the sequence GQLDW (SEQ ID NO:8) occurs in mammalian12S- and 15S-lipoxygenases, GQYDW (SEQ ID NO:35) occurs in5S-lipoxygenases, and GQFDS (SEQ ID NO:28) occurs in the new human15S-lipoxygenase, 15-Lox-2. The primer sequences are the same as thosedescribed in Example 1 above, except for the new degenerate downstreamprimer encoding GQFDS (SEQ ID NO:28): 5′-CCA-AGC-GCA-SSA-RTC-RAA-YTG-NCC(where S, “strong”, encodes C or G) (SEQ ID NO:29). For the second roundnested PCR reaction, the upstream primer was retained as before[WLLAK-(AAA) (SEQ ID NO:6) or WLLAK-(AAG) (SEQ ID NO:7)], while thedownstream primer was changed in all reactions to encode the sequenceELQXWWR (SEQ ID NO:26). After the second round PCR, only the reactionthat originally used the WLLAK-(AAG) (SEQ ID NO:7) and GQFDS primers(SEQ ID NO:29) yielded a visible PCR product. This product was 500 bp insize. The first round PCR reaction was primed with cDNA from phorbolester-treated mouse epidermis, 1 ml per 50 ml PCR reaction (from a 50 mlcDNA synthesis using 20 mg total RNA), and using 10 mM Tris, pH 8.3, 50mM KCl, 3 mM MgCl₂ with 0.2 mM of each dNTP and 0.25 ml (1.25 units) ofAmpliTaq DNA polymerase (Perkin Elmer) in a Perkin Elmer 480thermocycler. After the addition of the cDNA at 80° C. (hot start), thePCR was programmed as follows: 94° C. for 2 min, 1 cycle; 50° C. for 1min, 72° C. for 1 min, 94° C. for 1 min, 30 cycles; 72° C. for 10 min, 1cycle, and then the block temperature was held at 4° C. The second roundreaction was primed with the equivalent of 0.1 ml of the first roundreaction products (added as a 10-fold dilution). The protocol was 94° C.for 2 min, 1 cycle; 58° C. for 1 min, 72° C. for 1 min, 94° C. for 1 minfor 30 cycles; the protocol was completed with one cycle at 72° C. for10 min, and then the block temperature was held at 4° C.

[0198] 3′-RACE and 5′-RACE

[0199] The 3′ sequence was obtained using established upstream sequence5′ G-AGC-TTT-GTC-TCT-GAA-ATA-GTC-AG 3′ (SEQ ID NO:30) against adownstream primer based on the adaptor-linked oligo-dT primer used forCDNA synthesis (Brash et al. (1996) J. Biol. Chem. 271:20949-20957). The5′ RACE was accomplished using a kit from GIBCO BRL according to themanufacturer's instructions. The gene-specific downstream primers were5′ GTG-AGG-AAT-CAA-TAG-CTT-GAA-GAG 3′ (SEQ ID NO:31), and 5′G-ATG-TGT-GAC-AGC-CTC-ATG-GAT-G 3′ (SEQ ID NO:32).

[0200] Full-length Clones Obtained by PCR

[0201] The upstream primer encoded the N-terminus with a HindIII siteadded at the 5′ end to facilitate subcloning: 5′C-AAG-CTT-AGG-AGG-ATG-GCG-AAA-TGC-AGG 3′ (SEQ ID NO:33), and thedownstream primer encoded the C-terminus of the protein with an added5′EcoRI site: 5′ G-GAA-TTC-ATG-TTA-GAT-GGA-GAC-ACT-GTT 3′ (SEQ IDNO:34). These two primers were is purified by HPLC with the DMTprotecting groups on (Brash et al. (1996) J. Biol. Chem.271:20949-20957). After deprotection they were used in PCR reactionswith a proof-reading mixture of Taq/Pwo DNA polymerase (Expand HighFidelity, Boehringer-Mannheim) according to the manufacturer'sinstructions. The reaction conditions were 94° C., 2 min, 1 cycle; 58°C. for 30 sec, 72° C. for 1 min 30 sec, 96° C. for 15 sec, 3 cycles; 68°C. for 2 min, 96° C. 15 sec, 30 cycles; 72° C. for 10 min, 1 cycle; holdat 4° C.

[0202] DNA Sequencing

[0203] cDNAs were sequenced using the Oncor Fidelity manual dideoxychain termination method or by automated sequencing on a ABI Prism 310Genetic analyzer and fluorescence-tagged dye terminator cycle sequencing(Perkin Elmer).

[0204] HPLC Analysis of Tipoxygenase Metabolism

[0205] The lipoxygenase metabolism of [1-¹⁴C]arachidonic acid or[1-¹⁴C]linoleic acid was evaluated essentially as described previously(Hughes et al. (1991) Biochim. Biophys. Acta 1081:347-354). Followingincubation with 100 mM of substrate, products were extracted using theBligh and Dyer procedure (Bligh et al. (1959) Can. J. Biochem. Physiol.37:911-917), and the extracts were analyzed by RP-HPLC, SP-HPLC andchiral column analysis (Brash et al. (1990) Methods Enzymol.187:187-192). The hydroperoxide products were reduced withtriphenylphosphine, methylated with diazomethane, purified by SP-HPLC,and then the stereochemistry was analyzed using a Chiralcel OD column.

[0206] Expression of Mouse 8s-lipoxygenase Clones

[0207] The PCR products corresponding to the open reading frame of thecDNA were ligated directly into pCR3.1 (Invitrogen), and expressed bytransient transfection in HeLa cells using VTF-7, a recombinant vacciniavirus containing the T7 RNA polymerase gene (Blakely et al. (1991) Anal.Biochem. 194:302-308), or in human embryonic kidney (HEK) 293 cellsusing the adenovirus VA RNA gene. Funk et al. (1996) J. Biol. Chem.271:23338-23344. In the former system, cells plated at 1×10⁶ cells/35 mmwell 48 h earlier were transfected with 1 mg of plasmid DNA and 3 mg oflipofectin, and harvested after 12 hours. In the HEK system, cellsplated at 1×10⁶ cells/10 cm dish 24 h earlier were transfected with 10mg of plasmid DNA by the calcium phosphate method, and harvested aftertwo to three (2-3) days. Funk et al. (1996) J. Biol. Chem.271:23338-23344. The harvested cells were sonicated on ice, and theresulting homogenates were incubated with 100 mM [1-¹⁴C]arachidonic acidor [1-¹⁴C]linoleic acid for 45 min at room temperature. The metaboliteswere extracted and analyzed as described above.

[0208] Screening of cDNA Library

[0209] The library was a commercial 1 Unizap.XR skin cDNA libraryprepared using poly(A)+RNA isolated from whole skin of C57/Black femalemice (Stratagene). It was screened with a 347 bp BsaMI fragment of themouse 8S-lipoxygenase cDNA (PCR clone) as probe.

[0210] Northern Analysis

[0211] Poly(A)+RNA was prepared from PMA- or acetone-treated frozendorsal skin using TRI REAGENT® (Molecular Research Center, Inc.) andOligotex™ (Qiagen) according to the manufacturers' instructions. Thepoly(A)⁺ RNA was electrophoresed in 1% agarose/formaldehyde gel andblotted to a Hybond-H⁺ nylon membrane (Amersham). The membrane washybridized with ³²P-labeled DNA probe (complementary with a 0.6-kbEcoRV/BamHI fragment of mouse epidermal 8S-lipoxygenase) prepared usingthe Multiprime DNA labeling kit and Rapid-hybridization buffer(Amersham), and then washed according to the manufacturer'sspecifications. Blots were exposed to Fuji x-ray film at −80° C. Mousecyclophilin cDNA was used as a house-keeping gene to access loading ofRNA.

[0212] Western Analysis

[0213] After quantitation by Bradford assay (Bio-Rad), protein wasseparated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and thentransferred electrophoretically to Hybond ECL nitrocellulose membranes(Amersham). These were probed using a rabbit polyclonal antibody raisedagainst the human 15-Lox-2. This antibody recognises 15-Lox-2 and themouse 8S-lipoxygenase, but not the human reticulocyte type of15S-lipoxygenase, as more fully described below. Donkey anti-rabbit Iglinked with horseradish peroxidase (Amersham) was the secondaryantibody. Specifically bound protein was detected by chemiluminescenceusing the ECL Western blotting detection reagents (Amersham).

[0214] Immunohistochemical Analysis

[0215] The dorsal and tail skin of six to seven (6-7)-day-old mice weretreated with acetone or PMA (10 nmol for dorsal skin, 2 nmol for tailskin). After 24 h, the animals were euthanized and the dorsal and tailskin were washed with soap and then rinsed thoroughly with water. Wholedorsal and tail skin was immersion-fixed for 24 h in 4% paraformaldehydein phosphate-buffered saline (pH 7.4), dehydrated in ethanolic solutionsand xylenes, and embedded in paraffin. Skin sections weredeparaffinized, rehydrated in graded alcohols. Endogenous peroxidaseactivity was blocked in 3% hydrogen peroxide/methanol for 20 minfollowed by incubation in 10% goat serum for 20 min. Sections wereincubated at room temperature for 1 h in a {fraction (1/2500)} dilutionof either primary rabbit antisera that was used for Western analyses of8S-lipoxygenase or pre-immune sera. After rinsing in PBS, sections wereincubated with the biotinylated secondary antisera andperoxidase-labeled tertiary antisera supplied with an ABC Elite kit(Vector Corp, Guyrlingame, Calif.) followed by visualization ofimmunoprecipitate with DAB chromagen (Biogenix, Ban Ramon, Calif.).

[0216] Results of Molecular Cloning by RT-PCR

[0217] A series of PCR reactions were carried out with cDNA preparedfrom phorbol ester-treated mouse skin as template, and using degenerateprimers based on well conserved sequences in mammalian lipoxygenases.The primers were identical to those used in the cloning of a novel15S-lipoxygenase (15-Lox-2) from human skin as described in Example 1,with the addition of an extra downstream primer that better representedthe sequence of the new human enzyme. After running the protocol ofnested PCR reactions, a strong band of the expected size of 500 bp wasobtained in one of the reactions that used the new downstream primer(see above for details). The sequence of this PCR product showed astriking homology to the human lipoxygenase sequence. The remainder ofthe mouse cDNA was cloned by conventional 3′- and 5′-RACE. cDNAcorresponding to the open reading frame was prepared by PCR using aproof-reading mixture of Taq/Pwo as DNA polymerase. Eight of theseclones were selected for expression studies. Seven clones were fullysequenced.

[0218] Subsequently, a partial cDNA sequence was used to screen a mouseskin cDNA library and two full length cDNAs of 3.2 kb were isolated andsequenced. These were identical to each other and also matched exactlyone of the PCR products in the open reading frame (FIG. 5). FIG. 6 showsan alignment with the human 15-Lox-2. The sequences are 78% identical atboth the DNA and protein levels.

[0219] Transient Expression in HEK and Hela Cells

[0220] The library clone was obtained relatively late in this study, andtherefore much of the expression work described here was carried outusing several of the PCR products. It became apparent very early on thatthere is some problem in expression of this mouse lipoxygenase. Using astandard transient expression system in HEK 293 cells, it was only veryoccasionally that applicants detected enzymatic activity in theexpressed mouse lipoxygenase. Positive controls using the humanreticulocyte-type of 15S-lipoxygenase (15-Lox-1) or the second type ofhuman 15S-lipoxygenase (15-Lox-2) were run in every experiment, andthese cDNAs always expressed with readily detectible activity. In thefew instances when active mouse lipoxygenase was obtained in HEK cellexpression, the enzyme converted arachidonic acid to8S-hydroperoxyeicosatetraenoic acid (8S-HPETE).

[0221] Consistent expression of the mouse skin lipoxygenase was obtainedusing HeLa cells infected with vaccinia virus encoding the T7 RNApolymerase. Blakely et al. (1991) Anal. Biochem. 194:302-308. In thissystem, using sonicated cells from a 35 mm well, typically 30-40% ofadded arachidonic acid (100 μ∃M) was converted to 8S-HPETE as the soleenzymatic product (FIG. 7). The percentage conversion of arachidonicacid in this system was always similar to that obtained using the human15-Lox-2 as a positive control. Active enzyme was obtained using severalof the PCR clones (that encode one, two, or three different amino acidsfrom the library clone, FIG. 5 description) and the library cloneitself.

[0222] Using the vaccinia expression system, linoleic acid was found tobe a substrate for the mouse 8S-lipoxygenase although the conversion wastwo to three (2-3)-fold lower compared to arachidonic acid. The enzymeconverted linoleic acid exclusively to 9S-HODE (FIG. 8).

[0223] Effect of Phorbol Ester on Expression of 8S-lipoxygenase in MouseSkin

[0224] The expression level of 8S-lipoxygenase in mouse skin is known tobe strongly strain-dependent. Fischer et al. (1987) Cancer Res.47:3174-3179; Fischer et al. (1987) Carcinogenesis 8:421-424; Fischer etal. (1988) Cancer Res. 48:658-664. Also, the highest activity isinducible in six to ten (6-10)-day-old animals. Gschwendt et al. (1986)Carcinogenesis 7:449-455; Furstenberger et al. (1991) J. Biol. Chem.266:15738-15745. Applicants examined several strains of mice andobserved major differences in the level of constitutive expression (withno phorbol ester) and in the level after phorbol ester treatment. Forexample, using the Sencar strain applicants observed high constitutive8S-lipoxygenase activity in six to ten (6-10)-day-old pups, with littleextra induction by phorbol ester. The results shown here were obtainedusing a mixed breed of black Swiss animals that have low constitutiveactivity of 8S-lipoxygenase and exhibit strong induction with phorbolester. Using six to ten (6-10)-day-old pups, the inducing effect ofphorbol ester clearly is related to induction of both MRNA and protein(FIG. 9).

[0225] Cellular Localization of 8S-lipoxygenase in Mouse Skin

[0226] The localization of mouse 8S-lipoxygenase protein in dorsal skinand tail skin was observed and characterized by immunohistochemicalanalysis. A thickened hyperproliferative epidermis after PMA treatmentwas observed. An increase in 8S-lipoxygenase is due to an expansion incellularity in the stratum granulosum compartment of the epidermis.Baseline staining of the stratum granulosum for 8S-lipoxygenase in skinreceiving vehicle alone(acetone) was performed. The absence ofimmunoreactivity after incubation of PMA treated skin with pre-immuneantisera was also observed.

[0227] Thus, expression of the 8S-lipoxygenase protein was examined innormal mouse skin following treatment with phorbol ester in acetone(PMA) or acetone alone using the strain of black Swiss animalsresponsive to PMA. The histological analysis of skin from two differingbody locations (thin dorsal skin and thick tail skin) revealed a markedhyperproliferative response to PMA and a diminished response to theacetone vehicle alone. Most notable was an increase in the number ofdifferentiated cells within the outer epidermal compartment, the stratumgranulosum. The net result was more 8S-lipoxygenase positive cells inthe the PMA treated samples as compared to the samples receiving acetonealone. No immunoreactivity was detected in any samples reacted withpre-immune serum. Hair follicles positioned within the underlying dermisalso showed positive staining for 8S-lipoxygenase in differentiated celllayers. Staining in these locations did not show a modulation inresponse to topical treatment with phorbol ester.

[0228] Tissue Distribution of BS-lipoxygenase

[0229] As the related human 15S-lipoxygenase, 15-Lox-2, is expressed inprostate, an activity assay was used (HPLC analysis of products formedfrom [1-¹⁴C]arachidonic acid) to examine for 8S-lipoxygenase activity inmouse prostate. Using young adult males of 8 weeks of age, high levelsof cyclooxygenase and 12S-lipoxygenase activities were found in theprostate, but no 8S-lipoxygenase products were detected. Occurrence ofthe 8S-lipoxygenase transcript was examined in several different tissuesby Northern analysis. This revealed expression of 8S-lipoxygenasetranscript in mouse brain, with no detectible expression in heart,spleen, lung, liver, skeletal muscle, kidney and testis (FIG. 10).

[0230] Mouse 8S-lipoxygenase cDNA was cloned by PCR using primersrelated to the characterized human 15S-lipoxygenase (15-Lox-2) alsodescribed herein. These two lipoxygenases have 78% amino acid identity,and the differences are mainly conservative substitutions. The twoenzymes have 30-45% identity to other mammalian lipoxygenases. Theprimary structure of the mouse 8S-lipoxygenase contains the absolutelyconserved iron-binding histidines of lipoxygenases and the C-terminalisoleucine that is also an iron ligand. Boyington et al. (1993) Science260:1482-1486; Minor et al. (1996) Biochemistry 35:10687-10701. Anotable feature of the mouse 8S-lipoxygenase primary structure is thepresence of a serine at amino acid position 558 as the putative 5th ironligand. Minor et al. (1996) Biochemistry 35:10687-10701. The equivalentresidue in all other lipoxygenases is either a histidine or asparagine,with the exception of the human 15-Lox-2 in which a serine is alsopresent. Based on the sequence similarity, the 8S-lipoxygenase is themouse homologue of the human 15-Lox-2.

[0231] Initially, there was difficulty in studying the mouse enzyme asreliable expression of active lipoxygenase could not be obtained using aconventional HEK cell system. Chen et al. (1995) J. Biol. Chem.270:17993-17999; Chen et al. (1994) J. Biol. Chem. 269:13979-13987. Theproblem was solved by use of the recombinant vaccinia virus in aco-transfection system in Hela cells. In this procedure, the cells areco-transfected with the plasmid cDNA and vaccinia virus encoding the T7RNA polymerase. The virus protein induces high level expression via theT7 promoter upstream of the lipoxygenase CDNA. The cells are harvestedafter 12 hours. In this system, the mouse enzyme expressed withequivalent activity to either the 15-Lox-1 or 15-Lox-2 positivecontrols. Each of these lipoxygenases was expressed at a much higherlevel in the viral infected Hela cells than in the other procedure usingthe HEK cells.

[0232] The Western results show clearly that HEK cells produce the8S-lipoxygenase protein, although at lower levels than the positivecontrols (FIG. 7C). Whether this is a transfection problem, or relatedto translation or protein stability is not resolved. A similar poorexpression of activity of the mouse epidermal-type of 12S-lipoxygenasewas found in HEK cells. Funk et al. (1996) J. Biol. Chem.271:23338-23344. The lipoxygenase proteins expressed in HEK and HeLacells were indistinguishable in size by Western analysis. The very samepreparations of 8S-lipoxygenase plasmid cDNAs that failed to expressactive 8S-lipoxygenase in HEK cells were expressed with good activity inthe vaccinia system. Changing the vector from pCR3 to pCDNA3 had noeffect on HEK cell expression. Extracts of Hela cells (+/− viralinfection or vector alone) did not restore catalytic activity to HEKcells transfected with 8S-lipoxygenase. Furthermore, changing thevaccinia virus system over to HEK cells failed to induce expression of8S-lipoxygenase, whereas a 15-Lox-2 positive control gave easilymeasureable 15S-lipoxygenase activity. HEK cells may lack some factorthat helps the effective expression of certain lipoxygenase enzymes.

[0233] Linoleic acid was converted with about 3-fold lower efficiencythan arachidonic acid by the 8S-lipoxygenase. It was, however,specifically oxygenated to 9S-HPODE and likely participates in thebiosynthesis of this product in vivo. Linoleic acid is an abundantpolyunsaturated fatty acid in mouse skin and thus, potentially, thissubstrate is available. Ziboh et al. (1988) Prog. Lipid Res. 27:81-105.Lehmann and colleagues noted that the levels of 8-HETE and 9-HODE inmouse skin tend to change in parallel. Both are strikingly elevated inmouse skin papillomas are lower than normal in skin carcinomas. Lehmannet al. (1992) Anal. Biochem. 204:158-170. The 9S chirality of theproduct from linoleic acid is one criterion that could be used to assessthe contribution of the 8S-lipoxygenase to formation of 9-HODE in mouseskin. The main cyclooxygenase product from linoleic acid is theenantiomeric 9R-HODE (Hamberg et al. (1980) Biochim. Biophys. Acta617:545-547), while non-enzymatic reactions would give racemic product.

[0234] Northern and Western analyses, as well as the activity assay,showed that PMA treatment strongly induced de novo synthesis of mouse8S-lipoxygenase in the dorsal skin of the outbred mice used in thisexperiment. The histochemical analyses further defined the effect ofPMA. Immunoreactive 8S-lipoxygenase protein was most prominent in alayer of differentiated epidermis, the stratum granulosum. The thicknessof this cell layer increased markedly following 24 hours of treatmentwith PMA. An increase in the number of cells that produce8S-lipoxygenase, therefore, is one of the causes of the increased8S-lipoxygenase activity induced by PMA.

[0235] In the Northern analysis using a multiple tissue blot,8S-lipoxygenase mRNA was detected clearly in brain, but not in the otherseven tissues examined. Both the stratum granulosum of the epidermis andthe neuronal tissues of the central nervous system were originallyderived from the same ectodermal layer in early embryonic development,and both represent highly differentiated cell types. Occurrence of the8S-lipoxygenase transcript in brain was unexpected aslipbxygenase-catalyzed formation of 8-HETE has not been reported inneuronal tissues. The negative reaction in liver is of interest inrelation to the reported activity of 8S-HETE as a strong activator ofthe peroxisome proliferator-activated receptor, PPAR-a. Yu et al. (1995)J. Biol. Chem. 270:23975-23983. In liver, there is the possibility ofsynthesis of 8-HETE via the microsomal cytochrome P-450 system, althoughin vitro this gives a nearly racemic 8-HETE product. Capdevila et al.(1986) Biochem. Biophys. Res. Commun. 141:1007-1011.

[0236] The absence of 8S-lipoxygenase signal in the Northern analysis ofliver, could, however, be related to the lack of induction in normaltissue. The same issue applies to the absence of detectible8S-lipoxygenase activity in normal mouse prostate from young adultmales. Although the human homologue of the mouse 8S-lipoxygenase,15-Lox-2, was readily detectible in human prostate, as described inExample 1, the pooled human sample would include tissue from oldersubjects, the majority of which are expected to exhibit benign prostatichyperplasia. Oesterling, J. E. (1996) Prostate 6:67-73. The induction of8S-lipoxygenase in mouse skin by phorbol ester certainly is thus astriking feature of this enzyme.

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1 36 2685 base pairs nucleic acid double unknown not provided 1CAGGCGTGTC CCAGGGGGAG CCCCGCTCTG CAGCCCTGTG CGCCGTAGAG AGCTGGACTT 60AGGCTGGCAG C ATG GCC GAG TTC AGG GTC AGG GTG TCC ACC GGA GAA GCC 110 MetAla Glu Phe Arg Val Arg Val Ser Thr Gly Glu Ala 1 5 10 TTC GGG GCT GGCACA TGG GAC AAA GTG TCT GTC AGC ATC GTG GGG ACC 158 Phe Gly Ala Gly ThrTrp Asp Lys Val Ser Val Ser Ile Val Gly Thr 15 20 25 CGG GGA GAG AGC CCCCCA CTG CCC CTG GAC AAT CTC GGC AAG GAG TTC 206 Arg Gly Glu Ser Pro ProLeu Pro Leu Asp Asn Leu Gly Lys Glu Phe 30 35 40 45 ACT GCG GGC GCT GAGGAG GAC TTC CAG GTG ACG CTC CCG GAG GAC GTA 254 Thr Ala Gly Ala Glu GluAsp Phe Gln Val Thr Leu Pro Glu Asp Val 50 55 60 GGC CGA GTG CTG CTG CTGCGC GTG CAC AAG GCG CCC CCA GTG CTG CCC 302 Gly Arg Val Leu Leu Leu ArgVal His Lys Ala Pro Pro Val Leu Pro 65 70 75 CTG CTG GGG CCC CTG GCC CCGGAT GCC TGG TTC TGC CGC TGG TTC CAG 350 Leu Leu Gly Pro Leu Ala Pro AspAla Trp Phe Cys Arg Trp Phe Gln 80 85 90 CTG ACA CCG CCG CGG GGC GGC CACCTC CTC TTC CCC TGC TAC CAG TGG 398 Leu Thr Pro Pro Arg Gly Gly His LeuLeu Phe Pro Cys Tyr Gln Trp 95 100 105 CTG GAG GGG GCG GGG ACC CTG GTGCTG CAG GAG GGT ACA GCC AAG GTG 446 Leu Glu Gly Ala Gly Thr Leu Val LeuGln Glu Gly Thr Ala Lys Val 110 115 120 125 TCC TGG GCA GAC CAC CAC CCTGTG CTC CAG CAA CAG CGC CAG GAG GAG 494 Ser Trp Ala Asp His His Pro ValLeu Gln Gln Gln Arg Gln Glu Glu 130 135 140 CTT CAG GCC CGG CAG GAG ATGTAC CAG TGG AAG GCT TAC AAC CCA GGT 542 Leu Gln Ala Arg Gln Glu Met TyrGln Trp Lys Ala Tyr Asn Pro Gly 145 150 155 TGG CCT CAC TGC CTG GAT GAAAAG ACA GTG GAA GAC TTG GAG CTC AAT 590 Trp Pro His Cys Leu Asp Glu LysThr Val Glu Asp Leu Glu Leu Asn 160 165 170 ATC AAA TAC TCC ACA GCC AAGAAT GCC AAC TTT TAT CTA CAA GCT GGC 638 Ile Lys Tyr Ser Thr Ala Lys AsnAla Asn Phe Tyr Leu Gln Ala Gly 175 180 185 TCT GCT TTT GCA GAG ATG AAAATC AAG GGG TTG CTG GAC CGC AAG GGG 686 Ser Ala Phe Ala Glu Met Lys IleLys Gly Leu Leu Asp Arg Lys Gly 190 195 200 205 CTC TGG AGG AGT CTG AATGAG ATG AAA AGG ATC TTC AAC TTC CGG AGG 734 Leu Trp Arg Ser Leu Asn GluMet Lys Arg Ile Phe Asn Phe Arg Arg 210 215 220 ACC CCA GCA GCT GAG CACGCA TTT GAG CAC TGG CAG GAG GAT GCC TTC 782 Thr Pro Ala Ala Glu His AlaPhe Glu His Trp Gln Glu Asp Ala Phe 225 230 235 TTC GCC TCC CAG TTC CTGAAT GGT CTC AAC CCT GTC CTG ATC CGC CGC 830 Phe Ala Ser Gln Phe Leu AsnGly Leu Asn Pro Val Leu Ile Arg Arg 240 245 250 TGT CAC TAC CTC CCA AAGAAC TTC CCC GTC ACT GAT GCC ATG GTG GCC 878 Cys His Tyr Leu Pro Lys AsnPhe Pro Val Thr Asp Ala Met Val Ala 255 260 265 TCA TTG TTG GGT CCT GGGACC AGC TTG CAG GCT GAG CTA GAG AAG GGC 926 Ser Leu Leu Gly Pro Gly ThrSer Leu Gln Ala Glu Leu Glu Lys Gly 270 275 280 285 TCC CTG TTC TTG GTGGAT CAC GGC ATC CTC TCT GGC ATC CAG ACC AAT 974 Ser Leu Phe Leu Val AspHis Gly Ile Leu Ser Gly Ile Gln Thr Asn 290 295 300 GTC ATT AAT GGG AAGCCG CAG TTC TCT GCG GCC CCA ATG ACC CTG CTA 1022 Val Ile Asn Gly Lys ProGln Phe Ser Ala Ala Pro Met Thr Leu Leu 305 310 315 TAC CAG AGC CCA GGCTGC GGG CCG CTG CTG CCT CTC GCC ATC CAG CTC 1070 Tyr Gln Ser Pro Gly CysGly Pro Leu Leu Pro Leu Ala Ile Gln Leu 320 325 330 AGC CAG ACC CCC GGCCCA AAC AGC CCC ATC TTC CTG CCC ACT GAT GAC 1118 Ser Gln Thr Pro Gly ProAsn Ser Pro Ile Phe Leu Pro Thr Asp Asp 335 340 345 AAG TGG GAC TGG TTGCTG GCC AAG ACC TGG GTG CGC AAT GCC GAG TTC 1166 Lys Trp Asp Trp Leu LeuAla Lys Thr Trp Val Arg Asn Ala Glu Phe 350 355 360 365 TCC TTC CAT GAGGCC CTC ACG CAC CTG CTG CAC TCA CAT CTG CTG CCT 1214 Ser Phe His Glu AlaLeu Thr His Leu Leu His Ser His Leu Leu Pro 370 375 380 GAG GTC TTC ACCCTG GCT ACC CTG CGT CAG CTG CCC CAC TGC CAC CCT 1262 Glu Val Phe Thr LeuAla Thr Leu Arg Gln Leu Pro His Cys His Pro 385 390 395 CTC TTC AAG CTGCTG ATC CCG CAC ACC CGA TAC ACC CTG CAC ATC AAC 1310 Leu Phe Lys Leu LeuIle Pro His Thr Arg Tyr Thr Leu His Ile Asn 400 405 410 ACA CTC GCC CGGGAG CTG CTT ATC GTG CCA GGG CAG GTG GTG GAC AGG 1358 Thr Leu Ala Arg GluLeu Leu Ile Val Pro Gly Gln Val Val Asp Arg 415 420 425 TCC ACA GGC ATCGGC ATT GAA GGC TTC TCT GAG TTG ATA CAG AGG AAC 1406 Ser Thr Gly Ile GlyIle Glu Gly Phe Ser Glu Leu Ile Gln Arg Asn 430 435 440 445 ATG AAG CAGCTG AAC TAT TCT CTC CTG TGT CTG CCT GAG GAT ATC CGG 1454 Met Lys Gln LeuAsn Tyr Ser Leu Leu Cys Leu Pro Glu Asp Ile Arg 450 455 460 ACC CGA GGAGTT GAA GAC ATC CCA GGC TAC TAC TAC CGT GAT GAT GGG 1502 Thr Arg Gly ValGlu Asp Ile Pro Gly Tyr Tyr Tyr Arg Asp Asp Gly 465 470 475 ATG CAG ATTTGG GGT GCA GTG GAA CGC TTT GTC TCT GAA ATC ATC GGT 1550 Met Gln Ile TrpGly Ala Val Glu Arg Phe Val Ser Glu Ile Ile Gly 480 485 490 ATC TAC TACCCA AGT GAT GAG TCT GTC CAA GAT GAC AGA GAG CTC CAG 1598 Ile Tyr Tyr ProSer Asp Glu Ser Val Gln Asp Asp Arg Glu Leu Gln 495 500 505 GCC TGG GTCAGA GAG ATC TTC TCC AAG GGC TTC CTA AAC CAG GAG AGC 1646 Ala Trp Val ArgGlu Ile Phe Ser Lys Gly Phe Leu Asn Gln Glu Ser 510 515 520 525 TCA GGTATC CCT TCC TCA CTG GAG ACC CGG GAA GCC CTG GTG CAG TAT 1694 Ser Gly IlePro Ser Ser Leu Glu Thr Arg Glu Ala Leu Val Gln Tyr 530 535 540 GTC ACCATG GTG ATA TTC ACC TGC TCA GCC AAG CAT GCG GCT GTC AGT 1742 Val Thr MetVal Ile Phe Thr Cys Ser Ala Lys His Ala Ala Val Ser 545 550 555 GCA GGGCAG TTT GAC TCC TGT GCT TGG ATG CCC AAC CTG CCA CCC AGC 1790 Ala Gly GlnPhe Asp Ser Cys Ala Trp Met Pro Asn Leu Pro Pro Ser 560 565 570 ATG CAGCTG CCA CCA CCC ACC TCC AAA GGC CTG GCA ACA TGC GAG GGC 1838 Met Gln LeuPro Pro Pro Thr Ser Lys Gly Leu Ala Thr Cys Glu Gly 575 580 585 TTC ATAGCC ACC CTC CCA CCT GTC AAT GCC ACA TGT GAT GTC ATC CTT 1886 Phe Ile AlaThr Leu Pro Pro Val Asn Ala Thr Cys Asp Val Ile Leu 590 595 600 605 GCTCTC TGG TTG CTG AGC AAG GAG CCT GGA GAC CAA AGG CCC CTG GGC 1934 Ala LeuTrp Leu Leu Ser Lys Glu Pro Gly Asp Gln Arg Pro Leu Gly 610 615 620 ACCTAT CCG GAT GAG CAC TTC ACA GAG GAG GCC CCT CGG CGG AGC ATC 1982 Thr TyrPro Asp Glu His Phe Thr Glu Glu Ala Pro Arg Arg Ser Ile 625 630 635 GCCACC TTC CAG AGC CGC CTG GCC CAG ATC TCG AGG GGC ATC CAG GAG 2030 Ala ThrPhe Gln Ser Arg Leu Ala Gln Ile Ser Arg Gly Ile Gln Glu 640 645 650 CGGAAC CGG GGC CTG GTG CTG CCC TAC ACC TAC CTA GAC CCT CCC CTC 2078 Arg AsnArg Gly Leu Val Leu Pro Tyr Thr Tyr Leu Asp Pro Pro Leu 655 660 665 ATCGAG AAC AGC GTC TCC ATC TAAATCCCAG GGGAACACAG GCCCAGATGA 2129 Ile GluAsn Ser Val Ser Ile 670 675 CATCCCTTTG ACCACATCGC TCTAGGATAA CTGGCACCCAGAGAAAAGGA CTCCTCAGAA 2189 AAAACAGGCC CCCATGTGCC TCTCCTGGGA CAACCAGACTCTGTAACTCA CCCCCACCAC 2249 CATACACACA CACAAAAACA GAAACAAAAT CAAAACAGAGAAAGCAGAAA ATCTACCAAG 2309 AACAGAGTCT CAGGACAGAA CCACTGAGTC TTTTGGAGGCTCCAAGCCTC AAAGTGCCCG 2369 CAGAGCCCAC CTTGAGGGTT TTGCTAGTTG GTTTTGTTTTGCGTTTACAG CCGTGGGGGG 2429 AAGCACATAA TCCCGCCCCA GGGCCCACTA GCATCCACTGATTGGACCTT ATGGTCACCC 2489 AACTCAAGGA CAGCCACCAA GAAGTGGCTG CCAAAGAGACTGGGCGCAGT GGCTCATGCC 2549 CATAATCCCA GCACTTTGGG AGATGGAGGC GGGAAAATCATTTGAGGTCA GAAGTTCAAG 2609 GCCAGCCTGG ACGACATAGC GAGACTCCAC CTCTACCAAAAAATAAAAAT TAAAAAACAA 2669 AAAAAAAAAA AAAAAA 2685 676 amino acids aminoacid single unknown not provided 2 Met Ala Glu Phe Arg Val Arg Val SerThr Gly Glu Ala Phe Gly Ala 1 5 10 15 Gly Thr Trp Asp Lys Val Ser ValSer Ile Val Gly Thr Arg Gly Glu 20 25 30 Ser Pro Pro Leu Pro Leu Asp AsnLeu Gly Lys Glu Phe Thr Ala Gly 35 40 45 Ala Glu Glu Asp Phe Gln Val ThrLeu Pro Glu Asp Val Gly Arg Val 50 55 60 Leu Leu Leu Arg Val His Lys AlaPro Pro Val Leu Pro Leu Leu Gly 65 70 75 80 Pro Leu Ala Pro Asp Ala TrpPhe Cys Arg Trp Phe Gln Leu Thr Pro 85 90 95 Pro Arg Gly Gly His Leu LeuPhe Pro Cys Tyr Gln Trp Leu Glu Gly 100 105 110 Ala Gly Thr Leu Val LeuGln Glu Gly Thr Ala Lys Val Ser Trp Ala 115 120 125 Asp His His Pro ValLeu Gln Gln Gln Arg Gln Glu Glu Leu Gln Ala 130 135 140 Arg Gln Glu MetTyr Gln Trp Lys Ala Tyr Asn Pro Gly Trp Pro His 145 150 155 160 Cys LeuAsp Glu Lys Thr Val Glu Asp Leu Glu Leu Asn Ile Lys Tyr 165 170 175 SerThr Ala Lys Asn Ala Asn Phe Tyr Leu Gln Ala Gly Ser Ala Phe 180 185 190Ala Glu Met Lys Ile Lys Gly Leu Leu Asp Arg Lys Gly Leu Trp Arg 195 200205 Ser Leu Asn Glu Met Lys Arg Ile Phe Asn Phe Arg Arg Thr Pro Ala 210215 220 Ala Glu His Ala Phe Glu His Trp Gln Glu Asp Ala Phe Phe Ala Ser225 230 235 240 Gln Phe Leu Asn Gly Leu Asn Pro Val Leu Ile Arg Arg CysHis Tyr 245 250 255 Leu Pro Lys Asn Phe Pro Val Thr Asp Ala Met Val AlaSer Leu Leu 260 265 270 Gly Pro Gly Thr Ser Leu Gln Ala Glu Leu Glu LysGly Ser Leu Phe 275 280 285 Leu Val Asp His Gly Ile Leu Ser Gly Ile GlnThr Asn Val Ile Asn 290 295 300 Gly Lys Pro Gln Phe Ser Ala Ala Pro MetThr Leu Leu Tyr Gln Ser 305 310 315 320 Pro Gly Cys Gly Pro Leu Leu ProLeu Ala Ile Gln Leu Ser Gln Thr 325 330 335 Pro Gly Pro Asn Ser Pro IlePhe Leu Pro Thr Asp Asp Lys Trp Asp 340 345 350 Trp Leu Leu Ala Lys ThrTrp Val Arg Asn Ala Glu Phe Ser Phe His 355 360 365 Glu Ala Leu Thr HisLeu Leu His Ser His Leu Leu Pro Glu Val Phe 370 375 380 Thr Leu Ala ThrLeu Arg Gln Leu Pro His Cys His Pro Leu Phe Lys 385 390 395 400 Leu LeuIle Pro His Thr Arg Tyr Thr Leu His Ile Asn Thr Leu Ala 405 410 415 ArgGlu Leu Leu Ile Val Pro Gly Gln Val Val Asp Arg Ser Thr Gly 420 425 430Ile Gly Ile Glu Gly Phe Ser Glu Leu Ile Gln Arg Asn Met Lys Gln 435 440445 Leu Asn Tyr Ser Leu Leu Cys Leu Pro Glu Asp Ile Arg Thr Arg Gly 450455 460 Val Glu Asp Ile Pro Gly Tyr Tyr Tyr Arg Asp Asp Gly Met Gln Ile465 470 475 480 Trp Gly Ala Val Glu Arg Phe Val Ser Glu Ile Ile Gly IleTyr Tyr 485 490 495 Pro Ser Asp Glu Ser Val Gln Asp Asp Arg Glu Leu GlnAla Trp Val 500 505 510 Arg Glu Ile Phe Ser Lys Gly Phe Leu Asn Gln GluSer Ser Gly Ile 515 520 525 Pro Ser Ser Leu Glu Thr Arg Glu Ala Leu ValGln Tyr Val Thr Met 530 535 540 Val Ile Phe Thr Cys Ser Ala Lys His AlaAla Val Ser Ala Gly Gln 545 550 555 560 Phe Asp Ser Cys Ala Trp Met ProAsn Leu Pro Pro Ser Met Gln Leu 565 570 575 Pro Pro Pro Thr Ser Lys GlyLeu Ala Thr Cys Glu Gly Phe Ile Ala 580 585 590 Thr Leu Pro Pro Val AsnAla Thr Cys Asp Val Ile Leu Ala Leu Trp 595 600 605 Leu Leu Ser Lys GluPro Gly Asp Gln Arg Pro Leu Gly Thr Tyr Pro 610 615 620 Asp Glu His PheThr Glu Glu Ala Pro Arg Arg Ser Ile Ala Thr Phe 625 630 635 640 Gln SerArg Leu Ala Gln Ile Ser Arg Gly Ile Gln Glu Arg Asn Arg 645 650 655 GlyLeu Val Leu Pro Tyr Thr Tyr Leu Asp Pro Pro Leu Ile Glu Asn 660 665 670Ser Val Ser Ile 675 3205 base pairs nucleic acid double unknown notprovided 3 ATG GCG AAA TGC AGG GTG AGA GTA TCC ACG GGG GAA GCC TGT GGGGCT 48 Met Ala Lys Cys Arg Val Arg Val Ser Thr Gly Glu Ala Cys Gly Ala 15 10 15 GGC ACA TGG GAC AAA GTG TCT GTC AGC ATC GTG GGA ACC CAC GGA GAG96 Gly Thr Trp Asp Lys Val Ser Val Ser Ile Val Gly Thr His Gly Glu 20 2530 AGC CCC TTA GTA CCT CTG GAC CAT CTG GGC AAG GAG TTC AGC GCC GGT 144Ser Pro Leu Val Pro Leu Asp His Leu Gly Lys Glu Phe Ser Ala Gly 35 40 45GCT GAA GAA GAC TTC GAG GTG ACG CTT CCC CAG GAC GTA GGC ACT GTG 192 AlaGlu Glu Asp Phe Glu Val Thr Leu Pro Gln Asp Val Gly Thr Val 50 55 60 CTGATG CTG CGA GTC CAC AAA GCA CCC CCG GAA GTG TCC CTC CCG CTT 240 Leu MetLeu Arg Val His Lys Ala Pro Pro Glu Val Ser Leu Pro Leu 65 70 75 80 ATGTCT TTC CGT TCT GAT GCC TGG TTC TGC CGC TGG TTC GAG CTG GAG 288 Met SerPhe Arg Ser Asp Ala Trp Phe Cys Arg Trp Phe Glu Leu Glu 85 90 95 TGG CTACCT GGG GCT GCA CTC CAC TTC CCC TGT TAT CAG TGG CTG GAA 336 Trp Leu ProGly Ala Ala Leu His Phe Pro Cys Tyr Gln Trp Leu Glu 100 105 110 GGG GCGGGG GAG CTG GTG CTG AGA GAG GGA GCA GCA AAG GTG TCC TGG 384 Gly Ala GlyGlu Leu Val Leu Arg Glu Gly Ala Ala Lys Val Ser Trp 115 120 125 CAA GACCAT CAC CCT ACA CTG CAG GAT CAG CGC CAG AAG GAG CTT GAG 432 Gln Asp HisHis Pro Thr Leu Gln Asp Gln Arg Gln Lys Glu Leu Glu 130 135 140 TCC AGGCAG AAG ATG TAC AGC TGG AAG ACT TAC ATT GAA GGT TGG CCT 480 Ser Arg GlnLys Met Tyr Ser Trp Lys Thr Tyr Ile Glu Gly Trp Pro 145 150 155 160 CGCTGC CTT GAC CAC GAG ACT GTG AAA GAC TTG GAC CTC AAC ATC AAG 528 Arg CysLeu Asp His Glu Thr Val Lys Asp Leu Asp Leu Asn Ile Lys 165 170 175 TACTCT GCG ATG AAG AAT GCC AAA CTC TTC TTT AAA GCC CAC TCC GCG 576 Tyr SerAla Met Lys Asn Ala Lys Leu Phe Phe Lys Ala His Ser Ala 180 185 190 TATACG GAG CTG AAA GTC AAA GGG CTC CTG GAC CGC ACA GGA CTC TGG 624 Tyr ThrGlu Leu Lys Val Lys Gly Leu Leu Asp Arg Thr Gly Leu Trp 195 200 205 AGGAGT CTG AGG GAG ATG AGA AGG CTG TTT AAC TTC CGC AAG ACT CCA 672 Arg SerLeu Arg Glu Met Arg Arg Leu Phe Asn Phe Arg Lys Thr Pro 210 215 220 GCAGCA GAG TAT GTG TTT GCA CAC TGG CAG GAA GAT GCC TTC TTC GCC 720 Ala AlaGlu Tyr Val Phe Ala His Trp Gln Glu Asp Ala Phe Phe Ala 225 230 235 240TCC CAG TTC CTA AAT GGC ATC AAC CCG GTC CTG ATT CGC CGC TGT CAC 768 SerGln Phe Leu Asn Gly Ile Asn Pro Val Leu Ile Arg Arg Cys His 245 250 255AGT CTC CCA AAC AAC TTC CCG GTC ACT GAT GAA ATG GTG GCC CCA GTG 816 SerLeu Pro Asn Asn Phe Pro Val Thr Asp Glu Met Val Ala Pro Val 260 265 270CTG GGC CCT GGA ACC AGT CTG CAG GCT GAG TTG GAG AAG GGC TCC CTG 864 LeuGly Pro Gly Thr Ser Leu Gln Ala Glu Leu Glu Lys Gly Ser Leu 275 280 285TTC TTG GTG GAT CAT GGC ATT CTT TCT GGA GTC CAC ACC AAC ATC CTC 912 PheLeu Val Asp His Gly Ile Leu Ser Gly Val His Thr Asn Ile Leu 290 295 300AAT GGA AAG CCT CAG TTC TCT GCA GCC CCG ATG ACC CTG TTA CAC CAG 960 AsnGly Lys Pro Gln Phe Ser Ala Ala Pro Met Thr Leu Leu His Gln 305 310 315320 AGC TCA GGG TCC GGA CCC CTG CTT CCC ATT GCC ATC CAG CTC AAA CAG 1008Ser Ser Gly Ser Gly Pro Leu Leu Pro Ile Ala Ile Gln Leu Lys Gln 325 330335 ACT CCC GGG CCA GAC AAC CCC ATC TTC CTG CCC AGC GAT GAC ACG TGG 1056Thr Pro Gly Pro Asp Asn Pro Ile Phe Leu Pro Ser Asp Asp Thr Trp 340 345350 GAC TGG TTG CTG GCC AAG ACC TGG GTT CGC AAT TCT GAG TTT TAC ATC 1104Asp Trp Leu Leu Ala Lys Thr Trp Val Arg Asn Ser Glu Phe Tyr Ile 355 360365 CAT GAG GCT GTC ACA CAT CTG CTG CAT GCC CAT CTG ATT CCA GAA GTC 1152His Glu Ala Val Thr His Leu Leu His Ala His Leu Ile Pro Glu Val 370 375380 TTT GCC TTG GCC ACA TTA CGT CAG CTG CCT AGG TGT CAC CCT CTC TTC 1200Phe Ala Leu Ala Thr Leu Arg Gln Leu Pro Arg Cys His Pro Leu Phe 385 390395 400 AAG CTA TTG ATT CCT CAC ATT CGG TAC ACA CTG CAC ATC AAC ACG CTT1248 Lys Leu Leu Ile Pro His Ile Arg Tyr Thr Leu His Ile Asn Thr Leu 405410 415 GCC CGG GAG CTG CTC GTT GCC CCT GGG AAG TTG ATA GAC AAG TCC ACA1296 Ala Arg Glu Leu Leu Val Ala Pro Gly Lys Leu Ile Asp Lys Ser Thr 420425 430 GGC CTT GGC ACT GGG GGA TTC TCT GAC CTG ATA AAG AGA AAC ATG GAG1344 Gly Leu Gly Thr Gly Gly Phe Ser Asp Leu Ile Lys Arg Asn Met Glu 435440 445 CAG CTG AAC TAC TCT GTC CTG TGT CTC CCT GAA GAT ATC CGA GCC CGA1392 Gln Leu Asn Tyr Ser Val Leu Cys Leu Pro Glu Asp Ile Arg Ala Arg 450455 460 GGT GTG GAA GAC ATC CCA GGC TAC TAT TAC CGA GAT GAT GGG ATG CAG1440 Gly Val Glu Asp Ile Pro Gly Tyr Tyr Tyr Arg Asp Asp Gly Met Gln 465470 475 480 ATC TGG GGG GCA ATA AAG AGC TTT GTC TCT GAA ATA GTC AGC ATCTAC 1488 Ile Trp Gly Ala Ile Lys Ser Phe Val Ser Glu Ile Val Ser Ile Tyr485 490 495 TAT CCA AGT GAC ACA TCC GTC CAA GAT GAC CAA GAG CTC CAG GCCTGG 1536 Tyr Pro Ser Asp Thr Ser Val Gln Asp Asp Gln Glu Leu Gln Ala Trp500 505 510 GTG AGG GAG ATC TTC TCT GAG GGC TTC CTC GGC CGA GAA AGC TCAGGT 1584 Val Arg Glu Ile Phe Ser Glu Gly Phe Leu Gly Arg Glu Ser Ser Gly515 520 525 ATG CCC TCC TTG TTG GAT ACC CGG GAA GCC CTG GTC CAG TAT ATCACC 1632 Met Pro Ser Leu Leu Asp Thr Arg Glu Ala Leu Val Gln Tyr Ile Thr530 535 540 ATG GTG ATA TTC ACC TGC TCA GCC AAG CAT GCA GCT GTC AGT TCAGGC 1680 Met Val Ile Phe Thr Cys Ser Ala Lys His Ala Ala Val Ser Ser Gly545 550 555 560 CAG TTC GAC TCT TGT GTT TGG ATG CCC AAT CTG CCA CCT ACCATG CAG 1728 Gln Phe Asp Ser Cys Val Trp Met Pro Asn Leu Pro Pro Thr MetGln 565 570 575 CTA CCA CCA CCT ACT TCC AAA GGC CAG GCC CGG CCT GAG AGTTTC ATA 1776 Leu Pro Pro Pro Thr Ser Lys Gly Gln Ala Arg Pro Glu Ser PheIle 580 585 590 GCC ACG CTC CCA GCA GTT AAT TCG TCA AGT TAT CAC ATC ATTGCT CTC 1824 Ala Thr Leu Pro Ala Val Asn Ser Ser Ser Tyr His Ile Ile AlaLeu 595 600 605 TGG CTG CTA AGC GCA GAA CCT GGG GAC CAA AGG CCC CTG GGCCAC TAT 1872 Trp Leu Leu Ser Ala Glu Pro Gly Asp Gln Arg Pro Leu Gly HisTyr 610 615 620 CCA GAT GAA CAC TTC ACA GAG GAT GCC CCC CGG CGA AGC GTGGCT GCC 1920 Pro Asp Glu His Phe Thr Glu Asp Ala Pro Arg Arg Ser Val AlaAla 625 630 635 640 TTC CAG AGA AAG CTG ATC CAG ATC TCC AAG GGC ATC AGGGAG AGG AAC 1968 Phe Gln Arg Lys Leu Ile Gln Ile Ser Lys Gly Ile Arg GluArg Asn 645 650 655 CGA GGC CTG GCA CTG CCC TAC ACC TAC CTG GAT CCT CCCCTC ATT GAG 2016 Arg Gly Leu Ala Leu Pro Tyr Thr Tyr Leu Asp Pro Pro LeuIle Glu 660 665 670 AAC AGT GTC TCC ATC TAACATCTTG GAGAAGACAG TCCTGTGTGACATATAGAAC 2071 Asn Ser Val Ser Ile 675 TCTTGACCAT GCCTCTCCAG GCTAAGTCCCCGTATGCTTC TCCTGGACAA CCAAGCCCCA 2131 TCTTACACAC ACACACACAC ACACACACCTAATAAAATCG AAACAGAAAA ACCTAAACTC 2191 CCACAGAAGG CAAGATCTCA CACAGCAGAGAGCCATCCAA ATGTTTGGAG ACCCTGAGCT 2251 TCAGCTCTGA TTAACGGCTT TGCTGGTTTGCTTTGCTTTC TATTCCATTA ACCATGGACG 2311 GTAACAGAAA GCACAGAACC CTGGTTCACTGCACAAAGCC ACTGAGATCT CACCCTCACC 2371 TGACACAAAG GCAGCTATCA TACAGGCTTATCAGGAACAC AGGAATTTGT CCAATCAAAG 2431 CCTACCCACT AGGTCCATCG TGACCTACGACCTCACACTG GCATGCTTTA GCTTTGAGAA 2491 GGGATTACTG GAGTCAGGTA CGAAGAGAAGGACAGGACGA AGGCATGGCT CCATGTGGAA 2551 GAACATATCT GCTCTTCCAG ATGACCAGGGTAGCTCACAG CCATGTGTCA TTCTAACTCC 2611 AGAGGTCTCT AGTGGCCATG AAGACTCCAGGCATTCAGGG GATATACCAG TAGACACCAA 2671 AATTATACTT TTTAAGAGAG AGGATGGGCTGGAGAGATGG CTCAGCGGTT AAGAGCACTG 2731 ACTGCTCTTC CAGAGATCCT GAGTTCAATTCCCAGCAACC ACATGGTGGC TCACAACCAT 2791 CTGTAATGGG ATTCGATGCC CTCTTCTGGCGTGTCTGAAG ACAGCGACAG TGTATGCACA 2851 TATATAAAAT AAATAAATCT TTAAAAAACAAAACAAGAGA GAGGGACATG CTACCATTTC 2911 TACCTCACTT CTTCTCAAAG CCACCCCTAAAGTGAATTGT GAACCAGGTC CCCTTTGCAG 2971 AGAGTTAGAA GATATTCTCA AACCTCTAATACCTTCACAT CTAAAATCCA TCTTCATTCC 3031 AAAATTCCAA TATTTTATAT ACACTCTCCAGTTTGGTGGG TGAGGGGTTG TTTTTTGTTT 3091 GGTTTGGTTT GGTTGGGGTT TTGTTTTTGTTTTTGATTTT GTTTTTCTCT GGTTCAGACT 3151 CCATGGACGT TCATTAATGT CATAAATGAGTTCATTCCAA AAAAAAAAAA AAAA 3205 677 amino acids amino acid singleunknown not provided 4 Met Ala Lys Cys Arg Val Arg Val Ser Thr Gly GluAla Cys Gly Ala 1 5 10 15 Gly Thr Trp Asp Lys Val Ser Val Ser Ile ValGly Thr His Gly Glu 20 25 30 Ser Pro Leu Val Pro Leu Asp His Leu Gly LysGlu Phe Ser Ala Gly 35 40 45 Ala Glu Glu Asp Phe Glu Val Thr Leu Pro GlnAsp Val Gly Thr Val 50 55 60 Leu Met Leu Arg Val His Lys Ala Pro Pro GluVal Ser Leu Pro Leu 65 70 75 80 Met Ser Phe Arg Ser Asp Ala Trp Phe CysArg Trp Phe Glu Leu Glu 85 90 95 Trp Leu Pro Gly Ala Ala Leu His Phe ProCys Tyr Gln Trp Leu Glu 100 105 110 Gly Ala Gly Glu Leu Val Leu Arg GluGly Ala Ala Lys Val Ser Trp 115 120 125 Gln Asp His His Pro Thr Leu GlnAsp Gln Arg Gln Lys Glu Leu Glu 130 135 140 Ser Arg Gln Lys Met Tyr SerTrp Lys Thr Tyr Ile Glu Gly Trp Pro 145 150 155 160 Arg Cys Leu Asp HisGlu Thr Val Lys Asp Leu Asp Leu Asn Ile Lys 165 170 175 Tyr Ser Ala MetLys Asn Ala Lys Leu Phe Phe Lys Ala His Ser Ala 180 185 190 Tyr Thr GluLeu Lys Val Lys Gly Leu Leu Asp Arg Thr Gly Leu Trp 195 200 205 Arg SerLeu Arg Glu Met Arg Arg Leu Phe Asn Phe Arg Lys Thr Pro 210 215 220 AlaAla Glu Tyr Val Phe Ala His Trp Gln Glu Asp Ala Phe Phe Ala 225 230 235240 Ser Gln Phe Leu Asn Gly Ile Asn Pro Val Leu Ile Arg Arg Cys His 245250 255 Ser Leu Pro Asn Asn Phe Pro Val Thr Asp Glu Met Val Ala Pro Val260 265 270 Leu Gly Pro Gly Thr Ser Leu Gln Ala Glu Leu Glu Lys Gly SerLeu 275 280 285 Phe Leu Val Asp His Gly Ile Leu Ser Gly Val His Thr AsnIle Leu 290 295 300 Asn Gly Lys Pro Gln Phe Ser Ala Ala Pro Met Thr LeuLeu His Gln 305 310 315 320 Ser Ser Gly Ser Gly Pro Leu Leu Pro Ile AlaIle Gln Leu Lys Gln 325 330 335 Thr Pro Gly Pro Asp Asn Pro Ile Phe LeuPro Ser Asp Asp Thr Trp 340 345 350 Asp Trp Leu Leu Ala Lys Thr Trp ValArg Asn Ser Glu Phe Tyr Ile 355 360 365 His Glu Ala Val Thr His Leu LeuHis Ala His Leu Ile Pro Glu Val 370 375 380 Phe Ala Leu Ala Thr Leu ArgGln Leu Pro Arg Cys His Pro Leu Phe 385 390 395 400 Lys Leu Leu Ile ProHis Ile Arg Tyr Thr Leu His Ile Asn Thr Leu 405 410 415 Ala Arg Glu LeuLeu Val Ala Pro Gly Lys Leu Ile Asp Lys Ser Thr 420 425 430 Gly Leu GlyThr Gly Gly Phe Ser Asp Leu Ile Lys Arg Asn Met Glu 435 440 445 Gln LeuAsn Tyr Ser Val Leu Cys Leu Pro Glu Asp Ile Arg Ala Arg 450 455 460 GlyVal Glu Asp Ile Pro Gly Tyr Tyr Tyr Arg Asp Asp Gly Met Gln 465 470 475480 Ile Trp Gly Ala Ile Lys Ser Phe Val Ser Glu Ile Val Ser Ile Tyr 485490 495 Tyr Pro Ser Asp Thr Ser Val Gln Asp Asp Gln Glu Leu Gln Ala Trp500 505 510 Val Arg Glu Ile Phe Ser Glu Gly Phe Leu Gly Arg Glu Ser SerGly 515 520 525 Met Pro Ser Leu Leu Asp Thr Arg Glu Ala Leu Val Gln TyrIle Thr 530 535 540 Met Val Ile Phe Thr Cys Ser Ala Lys His Ala Ala ValSer Ser Gly 545 550 555 560 Gln Phe Asp Ser Cys Val Trp Met Pro Asn LeuPro Pro Thr Met Gln 565 570 575 Leu Pro Pro Pro Thr Ser Lys Gly Gln AlaArg Pro Glu Ser Phe Ile 580 585 590 Ala Thr Leu Pro Ala Val Asn Ser SerSer Tyr His Ile Ile Ala Leu 595 600 605 Trp Leu Leu Ser Ala Glu Pro GlyAsp Gln Arg Pro Leu Gly His Tyr 610 615 620 Pro Asp Glu His Phe Thr GluAsp Ala Pro Arg Arg Ser Val Ala Ala 625 630 635 640 Phe Gln Arg Lys LeuIle Gln Ile Ser Lys Gly Ile Arg Glu Arg Asn 645 650 655 Arg Gly Leu AlaLeu Pro Tyr Thr Tyr Leu Asp Pro Pro Leu Ile Glu 660 665 670 Asn Ser ValSer Ile 675 5 amino acids amino acid single linear not provided 5 TrpLeu Leu Ala Lys 1 5 21 base pairs nucleic acid single linear notprovided N=i=inosine 12, 15, 18 6 GACGTCTGGY TNYTNGCNAA A 21 21 basepairs nucleic acid single linear not provided N=i=inosine 12, 15, 18 7GACGTCTGGY TNYTNGCNAA G 21 5 amino acids amino acid single unknown notprovided 8 Gly Gln Leu Asp Trp 1 5 24 base pairs nucleic acid singlelinear not provided 9 CCAAGTGTAC CARTCNAGYT GNCC 24 24 base pairsnucleic acid single linear not provided 10 CCAAGTGTAC CARTCRTAYT GNCC 2434 base pairs nucleic acid single linear not provided N=i=inosine 24, 2511 TAGTCGACTG GCTTYTGGCC AAANNCTGGG TSCG 34 28 base pairs nucleic acidsingle linear not provided 12 GCGGATCCCT CCACCAGGNY TGSAGYTC 28 24 basepairs nucleic acid single linear not provided 13 GGTATCTACT ACCCAAGTGATGAG 24 21 base pairs nucleic acid single unknown not provided 14TACCCAAGTG ATGAGTCTGT C 21 23 base pairs nucleic acid single linear notprovided 15 GAAGACCTCA GGCAGCAGAT GTG 23 22 base pairs nucleic acidsingle linear not provided 16 TCATGGAAGG AGAACTCGGC AT 22 31 nucleicacid single linear not provided 17 ACGGATCCAG CATGGCCGAG TTCAGGGTCA G 3130 base pairs nucleic acid single linear not provided 18 CGGAATTCATGTCATCTGGG CCTGTGTTCC 30 19 base pairs nucleic acid single linear notprovided 19 TGCCTCTCGC CATCCAGCT 19 23 base pairs nucleic acid singlelinear not provided 20 TGTTCCCCTG GGATTTAGAT GGA 23 24 base pairsnucleic acid single linear not provided 21 GGTATCTACT ACCCAAGTGA TGAG 2421 base pairs nucleic acid single linear not provided 22 TGGGATGTCATCTGGGCCTG T 21 24 base pairs nucleic acid single linear not provided 23AACTCACCCC CACCACCATA CACA 24 23 base pairs nucleic acid single linearnot provided 24 TTCCCGCCTC CATCTCCCAA AGT 23 662 amino acids amino acidsingle unknown not provided 25 Met Gly Leu Tyr Arg Ile Arg Val Ser ThrGly Ala Ser Leu Tyr Ala 1 5 10 15 Gly Ser Asn Asn Gln Val Gln Leu TrpLeu Val Gly Gln His Gly Glu 20 25 30 Ala Ala Leu Gly Lys Arg Leu Trp ProAla Arg Gly Lys Glu Thr Glu 35 40 45 Leu Lys Val Glu Val Pro Glu Tyr LeuGly Pro Leu Leu Phe Val Lys 50 55 60 Leu Arg Lys Arg His Leu Leu Lys AspAsp Ala Trp Phe Cys Asn Trp 65 70 75 80 Ile Ser Val Gln Gly Pro Gly AlaGly Asp Glu Val Arg Phe Pro Cys 85 90 95 Tyr Arg Trp Val Glu Gly Asn GlyVal Leu Ser Leu Pro Glu Gly Thr 100 105 110 Gly Arg Thr Val Gly Glu AspPro Gln Gly Leu Phe Gln Lys His Arg 115 120 125 Glu Glu Glu Leu Glu GluArg Arg Lys Leu Tyr Arg Trp Gly Asn Trp 130 135 140 Lys Asp Gly Leu IleLeu Asn Met Ala Gly Ala Lys Leu Tyr Asp Leu 145 150 155 160 Pro Val AspGlu Arg Phe Leu Glu Asp Lys Arg Val Asp Phe Glu Val 165 170 175 Ser LeuAla Lys Gly Leu Ala Asp Leu Ala Ile Lys Asp Ser Leu Asn 180 185 190 ValLeu Thr Cys Trp Lys Asp Leu Asp Asp Phe Asn Arg Ile Phe Trp 195 200 205Cys Gly Gln Ser Lys Leu Ala Glu Arg Val Arg Asp Ser Trp Lys Glu 210 215220 Asp Ala Leu Phe Gly Tyr Gln Phe Leu Asn Gly Ala Asn Pro Val Val 225230 235 240 Leu Arg Arg Ser Ala His Leu Pro Ala Arg Leu Val Phe Pro ProGly 245 250 255 Met Glu Glu Leu Gln Ala Gln Leu Glu Lys Glu Leu Glu GlyGly Thr 260 265 270 Leu Phe Glu Ala Asp Phe Ser Leu Leu Asp Gly Ile LysAla Asn Val 275 280 285 Ile Leu Cys Ser Gln Gln His Leu Ala Ala Pro LeuVal Met Leu Lys 290 295 300 Leu Gln Pro Asp Gly Lys Leu Leu Pro Met ValIle Gln Leu Gln Leu 305 310 315 320 Pro Arg Thr Gly Ser Pro Pro Pro ProLeu Phe Leu Pro Thr Asp Pro 325 330 335 Pro Met Ala Trp Leu Leu Ala LysCys Trp Val Arg Ser Ser Asp Phe 340 345 350 Gln Leu His Glu Leu Gln SerHis Leu Leu Arg Gly His Leu Met Ala 355 360 365 Glu Val Ile Val Val AlaThr Met Arg Cys Leu Pro Ser Ile His Pro 370 375 380 Ile Phe Lys Leu IleIle Pro His Leu Arg Tyr Thr Leu Glu Ile Asn 385 390 395 400 Val Arg AlaArg Thr Gly Leu Val Ser Asp Met Gly Ile Phe Asp Gln 405 410 415 Ile MetSer Thr Gly Gly Gly Gly His Val Gln Leu Leu Lys Gln Ala 420 425 430 GlyAla Phe Leu Thr Tyr Ser Ser Phe Cys Pro Pro Asp Asp Leu Ala 435 440 445Asp Arg Gly Leu Leu Gly Val Lys Ser Ser Phe Tyr Ala Gln Asp Ala 450 455460 Leu Arg Leu Trp Glu Ile Ile Tyr Arg Tyr Val Glu Gly Ile Val Ser 465470 475 480 Leu His Tyr Lys Thr Asp Val Ala Val Lys Asp Asp Pro Glu LeuGln 485 490 495 Thr Trp Cys Arg Glu Ile Thr Glu Ile Gly Leu Gln Gly AlaGln Asp 500 505 510 Arg Gly Phe Pro Val Ser Leu Gln Ala Arg Asp Gln ValCys His Phe 515 520 525 Val Thr Met Cys Ile Phe Thr Cys Thr Gly Gln HisAla Ser Val His 530 535 540 Leu Gly Gln Leu Asp Trp Tyr Ser Trp Val ProAsn Ala Pro Cys Thr 545 550 555 560 Met Arg Leu Pro Pro Pro Thr Thr LysAsp Ala Thr Leu Glu Thr Val 565 570 575 Met Ala Thr Leu Pro Asn Phe HisGln Ala Ser Leu Gln Met Ser Ile 580 585 590 Thr Trp Gln Leu Gly Arg ArgGln Pro Val Met Val Ala Val Gly Gln 595 600 605 His Glu Glu Glu Tyr PheSer Gly Pro Glu Pro Lys Ala Val Leu Lys 610 615 620 Lys Phe Arg Glu GluLeu Ala Ala Leu Asp Lys Glu Ile Glu Ile Arg 625 630 635 640 Asn Ala LysLeu Asp Met Pro Tyr Glu Tyr Leu Arg Pro Ser Val Val 645 650 655 Glu AsnSer Val Ala Ile 660 7 amino acids amino acid single unknown not provided26 Glu Leu Gln Xaa Trp Trp Tyr 1 5 7 amino acids amino acid singleunknown not provided 27 Asp Val Trp Leu Leu Ala Lys 1 5 5 amino acidsamino acid single unknown not provided 28 Gly Gln Phe Asp Ser 1 5 24base pairs nucleic acid single linear not provided 29 CCAAGCGCASSARTCRAAYT GNCC 24 24 base pairs nucleic acid single linear not provided30 GAGCTTTGTC TCTGAAATAG TCAG 24 24 base pairs nucleic acid singlelinear not provided 31 GTGAGGAATC AATAGCTTGA AGAG 24 23 base pairsnucleic acid single linear not provided 32 GATGTGTGAC AGCCTCATGG ATG 2328 base pairs nucleic acid single linear not provided 33 CAAGCTTAGGAGGATGGCGA AATGCAGG 28 28 base pairs nucleic acid single linear notprovided 34 GGAATTCATG TTAGATGGAG ACACTGTT 28 5 amino acids amino acidsingle unknown not provided 35 Gly Gln Tyr Asp Trp 1 5 13 amino acidsamino acid single unknown not provided 36 Xaa Val Asp Trp Leu Leu AlaAla Lys Xaa Trp Val Arg 1 5 10

What is claimed is:
 1. A nucleic acid segment comprising an isolatedgene encoding a lipoxygenase, said lipoxygenase containing an ironligand comprising a serine.
 2. The nucleic acid segment of claim 1 ,wherein said isolated gene encodes a polypeptide having an in vivomolecular weight of about 76 KD when measured by SDS-PAGE.
 3. Thenucleic acid segment of claim 1 , wherein the encoded lipoxygenaseconverts arachidonic acid exclusively to 15S-hydroperoxyeicosatetraenoicacid or converts arachidonic acid exclusively to8S-hydroperoxyeicosatetraenoic acid.
 4. The nucleic acid segment ofclaim 1 , wherein the isolated gene encodes 15-Lox-2 or 8-Lox.
 5. Thenucleic acid segment of claim 1 , further defined as a DNA segment.
 6. Arecombinant host cell comprising the nucleic acid segment of claim 1 .7. The nucleic acid segment of claim 4 , wherein the isolated geneencodes 15-Lox-2.
 8. The nucleic acid segment of claim 4 , wherein theisolated gene encodes 8-Lox.
 9. The nucleic acid segment of claim 7 ,wherein the isolated gene encodes 15-Lox-2 comprising the amino acidsequence of SEQ ID NO:2.
 10. The nucleic acid segment of claim 9 ,further defined as comprising the 15-Lox-2-coding nucleic acid sequenceof SEQ ID NO:1.
 11. The nucleic acid segment of claim 8 , wherein theisolated gene encodes 8-Lox comprising the amino acid sequence of SEQ IDNO:4.
 12. The nucleic acid segment of claim 11 , further defined ascomprising 8-Lox-coding nucleic acid sequence of SEQ ID NO:3.
 13. Thenucleic acid segment of claim 5 , wherein the isolated gene ispositioned under the control of a promoter.
 14. The nucleic acid segmentof claim 13 , further defined as a recombinant vector which comprisesthe isolated gene.
 15. The nucleic acid segment of claim 14 , whereinthe vector is a recombinant expression vector.
 16. The recombinant hostcell of claim 6 , wherein the host cell is a procaryotic cell.
 17. Therecombinant host cell of claim 6 , wherein the host cell is a eucaryoticcell.
 18. A nucleic acid segment which comprises at least a 10nucleotide long contiguous stretch of the nucleic acid sequence of SEQID NO:1 or SEQ ID NO:3.
 19. The nucleic acid segment of claim 18 ,further defined as comprising at least a 15 nucleotide long contiguousstretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3. 20.The nucleic acid segment of claim 19 , further defined as comprising atleast a 20 nucleotide long contiguous stretch of the nucleic acidsequence of SEQ ID NO:1 or SEQ ID NO:3.
 21. The nucleic acid segment ofclaim 19 , further defined as a nucleic acid fragment of up to 10,000basepairs in length.
 22. The nucleic acid segment of claim 20 , furtherdefined as comprising at least a 30 nucleotide long contiguous stretchof the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 23. Thenucleic acid segment of claim 22 , further defined as comprising atleast a 50 nucleotide long contiguous stretch of the nucleic acidsequence of SEQ ID NO:1 or SEQ ID NO:3.
 24. The nucleic acid segment ofclaim 23 , further defined as comprising at least a 100 nucleotide longcontiguous stretch of the nucleic acid sequence of SEQ ID NO:1 or SEQ IDNO:3.
 25. The nucleic acid segment of claim 24 , further defined ascomprising at least a 1000 nucleotide long contiguous stretch of thenucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
 26. The nucleicacid segment of claim 25 , further defined as having the nucleic acidsequence of SEQ ID NO:1 or SEQ ID NO:3.
 27. The nucleic acid segment ofclaim 21 , further defined as a nucleic acid fragment of up to 1,000basepairs in length.
 28. The nucleic acid segment of claim 27 , furtherdefined as a nucleic acid fragment of up to 500 basepairs in length. 29.The nucleic acid segment of claim 28 , further defined as a nucleic acidfragment of up to 50 basepairs in length.
 30. A method of preparing alipoxygenase polypeptide, comprising: transforming a cell with thenucleic acid of claim 1 to produce a lipoxygenase under conditionssuitable for the expression of said polypeptide.
 31. A process ofdetecting in a sample an RNA that encodes the lipoxygenase polypeptideencoded by the nucleic acid of claim 1 , said process comprising thesteps of: (a) contacting said sample under hybridizing conditions withthe nucleic acid segment of claim 1 to form a duplex; and (b) detectingthe presence of said duplex.
 32. An isolated and purified biologicallyactive lipoxygenase polypeptide capable of converting arachidonic acidexclusively to 15S-hydroperoxyeicosatetraenoic acid, said lipoxygenasecontaining an iron ligand comprising a serine.
 33. A polypeptide ofclaim 32 , wherein said polypeptide has an in vivo molecular weight ofabout 76 KD when measured by SDS-PAGE.
 34. A polypeptide of claim 32 ,further comprising an amino acid sequence W-L-L-A-K (SEQ ID NO:5) and anamino acid sequence G-Q-Y-D-W (SEQ ID NO:35), the amino acid sequenceW-L-L-A-K (SEQ ID NO:5) positioned upstream from the amino acid sequenceG-Q-Y-D-W (SEQ ID NO:35) along the polypeptide.
 35. A polypeptideaccording to claim 32 , wherein the polypeptide comprises a 15-Lox-2.36. A polypeptide according to claim 35 , wherein the 15-Lox-2 comprisesthe amino acid sequence of SEQ ID NO:2.
 37. A polypeptide according toclaim 32 , modified to be in detectably labeled form.
 38. An isolatedand purified antibody capable of specifically binding to the polypeptideof claim 32 .
 39. The antibody of claim 38 which is a monoclonalantibody.
 40. The antibody of claim 38 which is a polyclonal antibody.41. A hybridoma cell line which produces the monoclonal antibody ofclaim 39 .
 42. An isolated and purified antibody capable of neutralizingthe biological activity of the polypeptide of claim 32 .
 43. Theantibody of claim 42 which is a monoclonal antibody.
 44. The antibody ofclaim 42 which is a polyclonal antibody.
 45. A hybridoma cell line whichproduces the monoclonal antibody of claim 43 .
 46. A process ofproducing an antibody immunoreactive with a lipoxygenase polypeptide,the process comprising steps of (a) transfecting a recombinant host cellwith the a polynucleotide of claim 1 , which encodes a lipoxygenasepolypeptide; (b) culturing the host cell under conditions sufficient forexpression of the polypeptide; (c) recovering the polypeptide; and (d)preparing the antibody to the polypeptide.
 47. The process of claim 46 ,wherein the polypeptide comprises SEQ ID NO:2.
 48. The process of claim46 , wherein the poynucleotide comprises SEQ ID NO:1 or comprises SEQ IDNO:3.
 49. An antibody produced by the process of claim 46 .
 50. Aprocess of detecting a lipoxygenase poypeptide, the process comprisingimmunoreacting the polypeptide with an antibody prepared according theprocess of claim 46 to form an antibody-polypeptide conjugate, anddetecting the conjugate.
 51. A process of detecting a messenger RNAtranscript that encodes a lipoxygenase polypeptide, the processcomprising the steps of hybridizing the messenger RNA transcript withthe polynucleotide of claim 1 to form a duplex; and detecting theduplex.
 52. A process of detecting a DNA molecule that encodes alipoxygenase polypeptide, the process comprising the steps ofhybridizing DNA molecules with the polynucleotide of claim 1 to form aduplex; and detecting the duplex.
 53. A diagnostic assay kit fordetecting the presence of a lipoxygenase polypeptide in a biologicalsample, the kit comprising a first container containing a first antibodycapable of immunoreacting with a lipoxygenase polypeptide encoded by thepolynucleotide of claim 1 , wherein the first antibody is present in anamount sufficient to perform at least one assay.
 54. An assay kit ofclaim 53 , further comprising a second container containing a secondantibody that immunoreacts with the first antibody.
 55. An assay kit ofclaim 54 , wherein the first antibody and the second antibody comprisemonoclonal antibodies.
 56. An assay kit of claim 55 , wherein the firstantibody is affixed to a solid support.
 57. An assay kit of claim 55 ,wherein the first and second antibodies each comprise an indicator. 58.An assay kit of claim 57 , wherein the indicator is a radioactive labelor an enzyme.
 59. A diagnostic assay kit for detecting the presence, inbiological samples, of a lipoxygenase polypeptide, the kit comprising afirst container that contains a polynucleotide identical orcomplimentary to a segment of at least ten contiguous nucleotide basesof the polynucleotide of claim 1 .
 60. A diagnostic assay kit fordetecting the presence, in a biological sample, of an antibodyimmunoreactive with a lipoxygenase polypeptide, the kit comprising afirst container containing a lipoxygenase polypeptide encoded by thepolynucleotide of claim 1 that immunoreacts with the antibody, with thepolypeptide present in an amount sufficient to perform at least oneassay.
 61. A screening assay for identifying a compound that affectsarachidonic acid metabolism in a cell, comprising the steps of: (a)establishing replicate test and control cultures of cells that express alipoxygenase polypeptide encoded by the polynucleotide of claim 1 ; (b)administering a candidate compound to the cells in the test culture butnot the control culture; (c) measuring hydroperoxyeicosatetraenoic acidlevels in the test and the control cultures; and (d) determining thatthe candidate compound affects arachidonic acid metabolism in a cell ifthe hydroperoxyeicosatetraenoic acid level measured for the test cultureis less or greater than the hydroperoxyeicosatetraenoic acid levelmeasured for the control culture.
 62. An assay of claim 61 , wherein thelipoxygenase polypeptide comprises 15-Lox-2.
 63. An assay of claim 61 ,wherein the lipoxygenase polypeptide comprises 8-Lox.